Nucleotide sequences mediating plant male fertility and method of using same

ABSTRACT

Nucleotide sequences mediating male fertility in plants are described, with DNA molecule and amino acid sequences set forth. Promoter sequences and their essential regions are also identified. The nucleotide sequences are useful in mediating male fertility in plants. In one such method, the homozygous recessive condition of male sterility causing alleles is maintained after crossing with a second plant, where the second plant contains a restoring transgene construct having a nucleotide sequence which reverses the homozygous condition. The restoring sequence is linked with a hemizygous sequence encoding a product inhibiting formation or function of male gametes. The maintainer plant produces only viable male gametes which do not contain the restoring transgene construct. Increase of the maintainer plant is also provided by self-fertilization, and selection for seed or plants which contain the construct.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of previously filed and co-pendingU.S. Ser. No. 12/400,578, filed Mar. 9, 2009, which is a divisionalapplication of previously filed U.S. Ser. No. 11/471,202, filed Jun. 20,2006, now U.S. Pat. No. 7,612,251, which is a continuation-in-part ofU.S. Ser. No. 11/166,609 filed Jun. 24, 2005, now U.S. Pat. No.7,517,975, which is a continuation-in-part of U.S. Ser. No. 10/412,000filed Apr. 11, 2003, now issued as U.S. Pat. No. 7,151,205, which is acontinuation of previously filed application U.S. Ser. No. 09/670,153,filed Sep. 26, 2000, now abandoned, all of which are incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Development of hybrid plant breeding has made possible considerableadvances in quality and quantity of crops produced. Increased yield andcombination of desirable characteristics, such as resistance to diseaseand insects, heat and drought tolerance, along with variations in plantcomposition are all possible because of hybridization procedures. Theseprocedures frequently rely heavily on providing for a male parentcontributing pollen to a female parent to produce the resulting hybrid.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinating if pollenfrom one flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

In Brassica, the plant is normally self-sterile and can only becross-pollinated. In self-pollinating species, such as soybeans andcotton, the male and female plants are anatomically juxtaposed. Duringnatural pollination, the male reproductive organs of a given flowerpollinate the female reproductive organs of the same flower.

Maize plants (Zea mays L.) present a unique situation in that they canbe bred by both self-pollination and cross-pollination techniques. Maizehas male flowers, located on the tassel, and female flowers, located onthe ear, on the same plant. It can self or cross pollinate. Naturalpollination occurs in maize when wind blows pollen from the tassels tothe silks that protrude from the tops of the incipient ears.

A reliable method of controlling fertility in plants would offer theopportunity for improved plant breeding. This is especially true fordevelopment of maize hybrids, which relies upon some sort of malesterility system and where a female sterility system would reduceproduction costs.

The development of maize hybrids requires the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Pedigree breeding and recurrent selection are two of thebreeding methods used to develop inbred lines from populations. Breedingprograms combine desirable traits from two or more inbred lines orvarious broad-based sources into breeding pools from which new inbredlines are developed by selfing and selection of desired phenotypes. Ahybrid maize variety is the cross of two such inbred lines, each ofwhich may have one or more desirable characteristics lacked by the otheror which complement the other. The new inbreds are crossed with otherinbred lines and the hybrids from these crosses are evaluated todetermine which have commercial potential. The hybrid progeny of thefirst generation is designated F₁. In the development of hybrids onlythe F₁ hybrid plants are sought. The F₁ hybrid is more vigorous than itsinbred parents. This hybrid vigor, or heterosis, can be manifested inmany ways, including increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility systemincorporating manual detasseling. To produce hybrid seed, the maletassel is removed from the growing female inbred parent, which can beplanted in various alternating row patterns with the male inbred parent.Consequently, providing that there is sufficient isolation from sourcesof foreign maize pollen, the ears of the female inbred will befertilized only with pollen from the male inbred. The resulting seed istherefore hybrid (F₁) and will form hybrid plants.

Environmental variation in plant development can result in plantstasseling after manual detasseling of the female parent is completed.Or, a detasseler might not completely remove the tassel of a femaleinbred plant. In any event, the result is that the female plant willsuccessfully shed pollen and some female plants will be self-pollinated.This will result in seed of the female inbred being harvested along withthe hybrid seed which is normally produced. Female inbred seed is not asproductive as F₁ seed. In addition, the presence of female inbred seedcan represent a germplasm security risk for the company producing thehybrid.

Alternatively, the female inbred can be mechanically detasseled bymachine. Mechanical detasseling is approximately as reliable as handdetasseling, but is faster and less costly. However, most detasselingmachines produce more damage to the plants than hand detasseling. Thus,no form of detasseling is presently entirely satisfactory, and a needcontinues to exist for alternatives which further reduce productioncosts and to eliminate self-pollination of the female parent in theproduction of hybrid seed.

A reliable system of genetic male sterility would provide advantages.The laborious detasseling process can be avoided in some genotypes byusing cytoplasmic male-sterile (CMS) inbreds. In the absence of afertility restorer gene, plants of a CMS inbred are male sterile as aresult of factors resulting from the cytoplasmic, as opposed to thenuclear, genome. Thus, this characteristic is inherited exclusivelythrough the female parent in maize plants, since only the femaleprovides cytoplasm to the fertilized seed. CMS plants are fertilizedwith pollen from another inbred that is not male-sterile. Pollen fromthe second inbred may or may not contribute genes that make the hybridplants male-fertile. Usually seed from detasseled normal maize and CMSproduced seed of the same hybrid must be blended to insure that adequatepollen loads are available for fertilization when the hybrid plants aregrown and to insure cytoplasmic diversity.

There can be other drawbacks to CMS. One is an historically observedassociation of a specific variant of CMS with susceptibility to certaincrop diseases. This problem has discouraged widespread use of that CMSvariant in producing hybrid maize and has had a negative impact on theuse of CMS in maize in general.

One type of genetic sterility is disclosed in U.S. Pat. Nos. 4,654,465and 4,727,219 to Brar, et al. However, this form of genetic malesterility requires maintenance of multiple mutant genes at separatelocations within the genome and requires a complex marker system totrack the genes and make use of the system convenient. Patterson alsodescribed a genic system of chromosomal translocations which can beeffective, but which are complicated. (See, U.S. Pat. Nos. 3,861,709 and3,710,511.)

Many other attempts have been made to improve on these drawbacks. Forexample, Fabijanski, et al., developed several methods of causing malesterility in plants (see EPO 89/3010153.8 publication no. 329,308 andPCT application PCT/CA90/00037 published as WO 90/08828). One methodincludes delivering into the plant a gene encoding a cytotoxic substanceassociated with a male tissue specific promoter. Another involves anantisense system in which a gene critical to fertility is identified andan antisense to the gene inserted in the plant. Mariani, et al. alsoshows several cytotoxic antisense systems. See EP 89/401, 194. Stillother systems use “repressor” genes which inhibit the expression ofanother gene critical to male sterility. PCT/GB90/00102, published as WO90/08829.

A still further improvement of this system is one described at U.S. Pat.No. 5,478,369 in which a method of imparting controllable male sterilityis achieved by silencing a gene native to the plant that is critical formale fertility and replacing the native DNA with the gene critical tomale fertility linked to an inducible promoter controlling expression ofthe gene. The plant is thus constitutively sterile, becoming fertileonly when the promoter is induced and its attached male fertility geneis expressed.

In a number of circumstances, a male sterility plant trait is expressedby maintenance of a homozygous recessive condition. Difficulties arisein maintaining the homozygous condition, when a transgenic restorationgene must be used for maintenance. For example, a natural mutation in agene critical to male sterility can impart a male sterility phenotype toplants when this mutant allele is in the homozygous state. Thissterility can be restored when the non-mutant form of the gene isintroduced into the plant. However, this form of restoration removes thedesired homozygous recessive condition, restores full male fertility andprevents maintenance of pure male sterile maternal lines. This issue canbe avoided where production of pollen containing the restoration gene iseliminated, thus providing a maintainer plant producing only pollen notcontaining the restoration gene, and the progeny retains the homozygouscondition. An example of one approach is shown in Dellaporta et al.,U.S. Pat. No. 6,743,968, in which a plant is produced having ahemizygotic construct comprising a gene that produces a product fatal toa cell, linked with a pollen-specific promoter, and the restorationgene. When crossed with the homozygous recessive male sterile plant, theprogeny thus retains the homozygous recessive condition.

As noted, an essential aspect of much of the work underway with malesterility systems is the identification of genes impacting malefertility.

Such a gene can be used in a variety of systems to control malefertility including those described herein. Previously, a male fertilitygene has been identified in Arabidopsis thaliana and used to produce amale sterile plant. Aarts, et al., “Transposon Tagging of a MaleSterility Gene in Arabidopsis”, Nature, 363:715-717 (Jun. 24, 1993).U.S. Pat. No. 5,478,369 discloses therein one such gene impacting malefertility. In the present invention the inventors provide novel DNAmolecules and the amino acid sequence encoded that are critical to malefertility in plants. These can be used in any of the systems wherecontrol of fertility is useful, including those described above.

Thus, one object of the invention is to provide a nucleic acid sequence,the expression of which is critical to male fertility in plants.

Another object of the invention is to provide a DNA molecule encoding anamino acid sequence, the expression of which is critical to malefertility in plants.

Yet another object of the invention is to provide a promoter of suchnucleotide sequence and its essential sequences.

A further object of the invention is to provide a method of using suchDNA molecules to mediate male fertility in plants.

Further objects of the invention will become apparent in the descriptionand claims that follow.

SUMMARY OF THE INVENTION

This invention relates to nucleic acid sequences, and, specifically, DNAmolecules and the amino acid encoded by the DNA molecules, which arecritical to male fertility. A promoter of the DNA is identified, as wellas its essential sequences. It also relates to use of such DNA moleculesto mediate fertility in plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a locus map of the male fertility gene Ms26.

FIG. 2A is a Southern blot of the ms26-m2::Mu8 family hybridized with aMu8 probe;

FIG. 2B is a Southern blot of the ms26-m2::Mu8 family hybridized with aPstI fragment isolated from the ms26 clone.

FIG. 3. is a Northern Blot analysis gel hybridized with a PstI fragmentisolated from the Ms26 gene.

FIG. 4A-4D is the sequence of Ms26 (The cDNA is SEQ ID NO: 1, theprotein is SEQ ID NOS: 2 and 34)

FIG. 5A-5C is the genomic Ms26 sequence (also referred to as SEQ ID NO:7).

FIG. 6A-6D is a comparison of the genomic Ms26 sequence (Residues1051-1450, 1501-2100 and 2201-3326 of SEQ ID NO: 7) with the cDNA ofMs26 (SEQ ID NO: 1).

FIG. 7A is a Northern analysis gel showing expression in various planttissues and FIG. 7B is a gel showing expression stages ofmicrosporogenesis

FIG. 8 is the full length promoter of Ms26 (SEQ ID NO: 5)

FIG. 9 is a bar graph showing luciferase activity after deletions ofselect regions of the Ms26 promoter.

FIG. 10 shows essential regions of the Ms26 promoter (SEQ ID NO: 6).

FIG. 11 is a bar graph showing luciferase activity after substitution byrestriction site linker scanning of select small (9-10 bp) regions ofthe Ms26 essential promoter fragment.

FIGS. 12A and 12B is a comparison of the nucleotide sequence (SEQ ID NO:3) from the Ms26 orthologue from a sorghum panicle and Ms26 maize cDNA(Residues 201-750 of SEQ ID NO: 1), and the sorghum protein sequence(SEQ ID NO: 4) and Ms26 maize protein (Residues 87-244 of SEQ ID NO: 2).

FIG. 13 is a representation of the mapping of the male sterility genems26.

FIG. 14 shows a sequence comparison of the region of excision of thems26-ref allele (SEQ ID NO: 8) with wild-type Ms26 (SEQ ID NO: 9).

FIG. 15 shows the transposon sequence within ms26-ref (SEQ ID NO: 10).

FIG. 16A-16B shows the entire ms26-ref sequence (SEQ ID NO: 11).

FIG. 17A shows a translated protein sequence alignment between regionsof the CYP704B1, a P450 gene (SEQ ID NO: 12) and Ms26 (SEQ ID NO: 13);FIG. 17B shows the phylogenetic tree analysis of select P450 genes.

FIG. 18 demonstrates the heme binding domain frame shift, showing thetranslated sequence alignment of regions of the Ms26 cDNA (SEQ ID NOS:14 and 28-29), the genomic regions of exon 5 in fertile plants (SEQ IDNOS: 15 and 30-31) and sterile plants (SEQ ID NOS: 16 and 32-33).

FIG. 19 shows the rice Ms26 cDNA (SEQ ID NO: 17) and protein (SEQ ID NO:18).

FIG. 20 shows alignment of the Ms26 promoter of corn (Residues 650-1091of SEQ ID NO: 5), sorghum (SEQ ID NO: 19) and rice (SEQ ID NO: 20).

FIG. 21 shows alignment of the maize Ms26 protein (SEQ ID NO: 21); riceMs26 protein (SEQ ID NO: 18) and sorghum Ms26 protein (SEQ ID NO: 22)along with a consensus sequence.

FIG. 22 is a plasmid map of PHP 18091, containing Ms45 fertility genewith a pollen promoter, cytotoxic gene and selectable marker.

FIG. 23 is a plasmid map of PHP 24101, containing the Ms26 fertilitygene with a pollen promoter, cytotoxic gene and selectable marker.

FIG. 24 shows a sequence of the Zea mays α-amylase 1 coding region (SEQID NOS: 26 (DNA) and 36 (protein)).

DISCLOSURE OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated therein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

Genetic male sterility results from a mutation, suppression, or otherimpact to one of the genes critical to a specific step inmicrosporogenesis, the term applied to the entire process of pollenformulation. These genes can be collectively referred to as malefertility genes (or, alternatively, male sterility genes). There aremany steps in the overall pathway where gene function impacts fertility.This seems aptly supported by the frequency of genetic male sterility inmaize. New alleles of male sterility mutants are uncovered in materialsthat range from elite inbreds to unadapted populations.

At U.S. Pat. No. 5,478,369 there is described a method by which the Ms45male sterility gene was tagged and cloned on maize chromosome 9.Previously, there had been described a male sterility gene on chromosome9, ms2, which had never been cloned and sequenced. It is not allelic tothe gene referred to in the '369 patent. See Albertsen, M. and Phillips,R. L., “Developmental Cytology of 13 Genetic Male Sterile Loci in Maize”Canadian Journal of Genetics & Cytology 23:195-208 (January 1981). Theonly fertility gene cloned before that had been the Arabadopsis genedescribed at Aarts, et al., supra.

Examples of genes that have been discovered subsequently that arecritical to male fertility are numerous and include the ArabidopsisABORTED MICROSPORES (AMS) gene, Sorensen et al., The Plant Journal(2003) 33(2):413-423); the Arabidopsis MS1 gene (Wilson et al., ThePlant Journal (2001) 39(2):170-181); the NEF1 gene (Ariizumi et al., ThePlant Journal (2004) 39(2):170-181); Arabidopsis AtGPAT1 gene (Zheng etal., The Plant Cell (2003) 15:1872-1887); the Arabdiopsis dde2-2mutation was shown to be defective in the allene oxide syntase gene(Malek et al., Planta (2002) 216:187-192); the Arabidopsis facelesspollen-1 gene (flp1) (Ariizumi et al, Plant Mol. Biol. (2003)53:107-116); the Arabidopisis MALE MEIOCYTE DEATH1 gene (Yang et al.,The Plant Cell (2003) 15: 1281-1295); the tapetum-specific zinc fingergene, TAZ1 (Kapoor et al., The Plant Cell (2002) 14:2353-2367); and theTAPETUM DETERMINANT1 gene (Lan et al, The Plant Cell (2003)15:2792-2804).

The table below lists a number of known male fertility mutants or genesfrom Zea mays.

GENE NAME ALTERNATE NAME REFERENCE ms1 male sterile1 male sterile1, ms1Singleton, WR and Jones, DF. 1930. J Hered 21: 266-268 ms10 malesterile10 male sterile10, ms10 Beadle, GW. 1932. Genetics 17: 413-431ms11 male sterile11 ms11, male sterile11 Beadle, GW. 1932. Genetics 17:413-431 ms12 male sterile12 ms12, male sterile12 Beadle, GW. 1932.Genetics 17: 413-431 ms13 male sterile13 ms*-6060, male sterile13,Beadle, GW. 1932. ms13 Genetics 17: 413-431 ms14 male sterile14 ms14,male sterile14 Beadle, GW. 1932. Genetics 17: 413-431 ms17 malesterile17 ms17, male sterile17 Emerson, RA. 1932. Science 75: 566 ms2male sterile2 male sterile2, ms2 Eyster, WH. 1931. J Hered 22: 99-102ms20 male sterile20 ms20, male sterile20 Eyster, WH. 1934. Genetics ofZea mays. Bibliographia Genetica 11: 187-392 ms23 male sterile23 :ms*-6059, ms*-6031, ms*- West, DP and Albertsen, 6027, ms*-6018,ms*-6011, MC. 1985. MNL 59: 87 ms35, male sterile23, ms*- Bear7, ms23ms24 male sterile24 ms24, male sterile24 West, DP and Albertsen, MC.1985. MNL 59: 87 ms25 male sterile25 ms*-6065, ms *-6057, Loukides, CA;Broadwater, ms25, male sterile25, ms*- AH; Bedinger, PA. 1995. 6022 Am JBot 82: 1017-1023 ms27 male sterile27 ms27, male sterile27 Albertsen,MC. 1996. MNL 70: 30-31 ms28 male sterile28 ms28, male sterile28Golubovskaya, IN. 1979. MNL 53: 66-70 ms29 male sterile29 malesterile29, ms*-JH84A, Trimnell, MR et al. 1998. ms29 MNL 72: 37-38 ms3male sterile3 Group 3, ms3, male sterile3 Eyster, WH. 1931. J Hered 22:99-102 ms30 male sterile30 ms30, msx, ms*-6028, ms*- Albertsen, MC etal. 1999. Li89, male sterile30, ms*- MNL 73: 48 LI89 ms31 male sterile31ms*-CG889D, ms31, male Trimnell, MR et al. 1998. sterile31 MNL 72: 38ms32 male sterile32 male sterile32, ms32 Trimnell, MR et al. 1999. MNL73: 48-49 ms33 male sterile33 : ms*-6054, ms*-6024, Patterson, EB. 1995.MNL ms33, ms*-GC89A, ms*- 69: 126-128 6029, male sterile6019, Group 7,ms*-6038, ms*- Stan1, ms*-6041, ms*- 6019, male sterile33 ms34 malesterile34 Group 1, ms*-6014, ms*- Patterson, EB. 1995. MNL 6010, malesterile34, ms34, 69: 126-128 ms*-6013, ms*-6004, male sterile6004 ms36male sterile36 male sterile36, ms*-MS85A, Trimnell, MR et al. 1999. ms36MNL 73: 49-50 ms37 male sterile 37 ms*-SB177, ms37, male Trimnell, MR etal. 1999. sterile 37 MNL 73: 48 ms38 male sterile38 ms30, ms38,ms*-WL87A, Albertsen, MC et al. 1996. male sterile38 MNL 70: 30 ms43male sterile43 ms43, male sterile43, ms29 Golubovskaya, IN. 1979. IntRev Cytol 58: 247-290 ms45 male sterile45 Group 6, male sterile45,Albertsen, MC; Fox, TW; ms*-6006, ms*-6040, ms*- Trimnell, MR. 1993.Proc BS1, ms*-BS2, ms*-BS3, Annu Corn Sorghum Ind ms45, ms45′-9301 ResConf 48: 224-233 ms48 male sterile48 male sterile48, ms*-6049, Trimnell,M et al. 2002. ms48 MNL 76: 38 ms5 male sterile5 : ms*-6061, ms*-6048,ms*- Beadle, GW. 1932. 6062, male sterile5, ms5 Genetics 17: 413-431ms50 male sterile50 ms50, male sterile50, ms*- Trimnell, M et al. 2002.6055, ms*-6026 MNL 76: 39 ms7 male sterile7 ms7, male sterile7 Beadle,GW. 1932. Genetics 17: 413-431 ms8 male sterile8 male sterile8, ms8Beadle, GW. 1932. Genetics 17: 413-431 ms9 male sterile9 Group 5, malesterile9, ms9 Beadle, GW. 1932. Genetics 17: 413-431 ms49 male sterile49ms*-MB92, ms49, male sterile49 Trimnell, M et al. 2002. MNL 76: 38-39

Thus the invention includes using the sequences shown herein to impactmale fertility in a plant, that is, to control male fertility bymanipulation of the genome using the genes of the invention. By way ofexample, without limitation, any of the methods described infra can beused with the sequence of the invention such as introducing a mutantsequence into a plant to cause sterility, causing mutation to the nativesequence, introducing an antisense of the sequence into the plant, useof hairpin formations, linking it with other sequences to control itsexpression, or any one of a myriad of processes available to one skilledin the art to impact male fertility in a plant.

The Ms26 gene described herein is located on maize chromosome 1 and itsdominant allele is critical to male fertility. The locus map isrepresented at FIG. 1. It can be used in the systems described above,and other systems impacting male fertility.

The maize family cosegregating for sterility was named ms*-SBMu200 andwas found to have an approximately 5.5 Kb EcoRI fragment that hybridizedwith a Mu8 probe (FIG. 2A). A genomic clone from the family was isolatedwhich contained a Mu8 transposon. A probe made from DNA bordering thetransposon was found to hybridize to the same ˜5.5 Kb EcoR1 fragment(FIG. 2B). This probe was used to isolate cDNA clones from a tassel cDNAlibrary. The cDNA is 1906 bp, and the Mu insertion occurred in exon 1 ofthe gene. This probe was also used to map the mutation in an RFLPmapping population. The mutant mapped to the short arm of chromosome 1,near Ms26. Allelism crosses between ms26-ref and ms*-SBMu200 showed thatthese were allelic, indicating that the mutations occurred in the samegene. The ms*-SBMu200 allele was renamed ms26-m2::Mu8. Two additionalalleles for the Ms26 gene were cloned, one containing a Mutator elementin the second exon, named ms26-m3::Mu*, and one containing an unknowntransposon in the fifth exon from the ms26-ref allele. FIG. 5 (discussedfurther below) represents the genomic nucleotide sequence. Expressionpatterns, as determined by Northern analysis, show tassel specificitywith peak expression at about the quartet to quartet release stages ofmicrosporogenesis.

It will be evident to one skilled in the art that variations, mutations,derivations including fragments smaller than the entire sequence setforth may be used which retain the male sterility controlling propertiesof the gene. One of ordinary skill in the art can readily assess thevariant or fragment by introduction into plants homozygous for a stablemale sterile allele of Ms26, followed by observation of the plant's maletissue development.

The sequences of the invention may be isolated from any plant,including, but not limited to corn (Zea mays), canola (Brassica napus,Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), millet (Panicum spp.), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats (Avena sativa), barley (Hordeum vulgare), vegetables,ornamentals, and conifers. Preferably, plants include corn, soybean,sunflower, safflower, canola, wheat, barley, rye, alfalfa, rice, cottonand sorghum.

Sequences from other plants may be isolated according to well-knowntechniques based on their sequence homology to the homologous codingregion of the coding sequences set forth herein. In these techniques,all or part of the known coding sequence is used as a probe whichselectively hybridizes to other sequences present in a population ofcloned genomic DNA fragments (i.e. genomic libraries) from a chosenorganism. Methods are readily available in the art for the hybridizationof nucleic acid sequences. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocolsin Molecular Biology, Chapter 2, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

Thus the invention also includes those nucleotide sequences whichselectively hybridize to the Ms26 nucleotide sequences under stringentconditions. In referring to a sequence that “selectively hybridizes”with Ms26, the term includes reference to hybridization, under stringenthybridization conditions, of a nucleic acid sequence to the specifiednucleic acid target sequence to a detectably greater degree than itshybridization to non-target nucleic acid.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences. Stringent conditions are target-sequence-dependent and willdiffer depending on the structure of the polynucleotide. By controllingthe stringency of the hybridization and/or washing conditions, targetsequences can be identified which are 100% complementary to a probe(homologous probing). Alternatively, stringency conditions can beadjusted to allow some mismatching in sequences so that lower degrees ofsimilarity are detected (heterologous probing). Generally, probes ofthis type are in a range of about 1000 nucleotides in length to about250 nucleotides in length.

An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). See also Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

In general, sequences that correspond to the nucleotide sequences of thepresent invention and hybridize to the nucleotide sequence disclosedherein will be at least 50% homologous, 70% homologous, and even 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%homologous or more with the disclosed sequence. That is, the sequencesimilarity between probe and target may range, sharing at least about50%, about 70%, and even about 85% or more sequence similarity.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. Generally, stringent wash temperature conditions areselected to be about 5° C. to about 2° C. lower than the melting point(Tm) for the specific sequence at a defined ionic strength and pH. Themelting point, or denaturation, of DNA occurs over a narrow temperaturerange and represents the disruption of the double helix into itscomplementary single strands. The process is described by thetemperature of the midpoint of transition, Tm, which is also called themelting temperature. Formulas are available in the art for thedetermination of melting temperatures.

Preferred hybridization conditions for the nucleotide sequence of theinvention include hybridization at 42° C. in 50% (w/v) formamide, 6×SSC,0.5% (w/v) SDS, 100(g/ml salmon sperm DNA. Exemplary low stringencywashing conditions include hybridization at 42° C. in a solution of2×SSC, 0.5% (w/v) SDS for 30 minutes and repeating. Exemplary moderatestringency conditions include a wash in 2×SSC, 0.5% (w/v) SDS at 50° C.for 30 minutes and repeating. Exemplary high stringency conditionsinclude a wash in 0.1×SSC, 0.1% (w/v) SDS, at 65° C. for 30 minutes toone hour and repeating. Sequences that correspond to the promoter of thepresent invention may be obtained using all the above conditions. Forpurposes of defining the invention, the high stringency conditions areused.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, or 100nucleotides in length, or longer. Those of skill in the art understandthat to avoid a high similarity to a reference sequence due to inclusionof gaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of aligning sequences for comparison are well-known in the art.Thus, the determination of percent sequence identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988)CABIOS 4: 11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2: 482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; thesearch-for-local-alignment-method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153;Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al.(1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller(1988) supra. A PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. The BLAST programs of Altschul et al (1990) J.Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul(1990) supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25: 3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3 and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2; and theBLOSUM62 scoring matrix or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen, GAP must, in addition, make a profit foreach gap inserted of the length of the gap times the gap extensionpenalty. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the GCG Wisconsin Genetics Software Package forprotein sequences are 8 and 2, respectively. For nucleotide sequencesthe default gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

Identity to the sequence of the present invention would mean apolynucleotide sequence having at least 65% sequence identity, morepreferably at least 70% sequence identity, more preferably at least 75%sequence identity, more preferably at least 80% identity, morepreferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity.

Promoter regions can be readily identified by one skilled in the art.The putative start codon containing the ATG motif is identified andupstream from the start codon is the presumptive promoter. By “promoter”is intended a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. A promoter can additionally comprise other recognitionsequences generally positioned upstream or 5′ to the TATA box, referredto as upstream promoter elements, which influence the transcriptioninitiation rate. It is recognized that having identified the nucleotidesequences for the promoter region disclosed herein, it is within thestate of the art to isolate and identify further regulatory elements inthe region upstream of the TATA box from the particular promoter regionidentified herein. Thus the promoter region disclosed herein isgenerally further defined by comprising upstream regulatory elementssuch as those responsible for tissue and temporal expression of thecoding sequence, enhancers and the like. In the same manner, thepromoter elements which enable expression in the desired tissue such asmale tissue can be identified, isolated, and used with other corepromoters to confirm male tissue-preferred expression. By core promoteris meant the minimal sequence required to initiate transcription, suchas the sequence called the TATA box which is common to promoters ingenes encoding proteins. Thus the upstream promoter of Ms26 canoptionally be used in conjunction with its own or core promoters fromother sources. The promoter may be native or non-native to the cell inwhich it is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the heterologousnucleotide sequence. Less than the entire promoter region can beutilized and the ability to drive anther-preferred expression retained.However, it is recognized that expression levels of mRNA can bedecreased with deletions of portions of the promoter sequence. Thus, thepromoter can be modified to be a weak or strong promoter. Generally, by“weak promoter” is intended a promoter that drives expression of acoding sequence at a low level. By “low level” is intended levels ofabout 1/10,000 transcripts to about 1/100,000 transcripts to about1/500,000 transcripts. Conversely, a strong promoter drives expressionof a coding sequence at a high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1,000 transcripts. Generally, atleast about 30 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence. It is recognized that toincrease transcription levels, enhancers can be utilized in combinationwith the promoter regions of the invention. Enhancers are nucleotidesequences that act to increase the expression of a promoter region.Enhancers are known in the art and include the SV40 enhancer region, the35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region of 5′ untranslation region (5′UTR) Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon. The term “isolated” refers to material such as anucleic acid or protein which is substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in it naturally occurring environment or if the material is in itsnatural environment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in a cell otherthan the locus native to the material. Methods for isolation of promoterregions are well known in the art.

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions.Functional variants of the invention may be created by site-directedmutagenesis, induced mutation, or may occur as allelic variants(polymorphisms).

As used herein, a “functional fragment” of the regulatory sequence is anucleotide sequence that is a regulatory sequence variant formed by oneor more deletions from a larger sequence. For example, the 5′ portion ofa promoter up to the TATA box near the transcription start site can bedeleted without abolishing promoter activity, as described byOpsahl-Sorteberg, H-G. et al., “Identification of a 49-bp fragment ofthe HvLTP2 promoter directing aleruone cell specific expression” Gene341:49-58 (2004). Such variants should retain promoter activity,particularly the ability to drive expression in male tissues. Activitycan be measured by Northern blot analysis, reporter activitymeasurements when using transcriptional fusions, and the like. See, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.),herein incorporated by reference.

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology See particularly, Mullis et al. (1987) Methods Enzymol.155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, NewYork).

Sequences which hybridize to the regulatory sequences of the presentinvention are within the scope of the invention. Sequences thatcorrespond to the promoter sequences of the present invention andhybridize to the promoter sequences disclosed herein will be at least50% homologous, 70% homologous, and even 85% 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous or more with thedisclosed sequence.

Smaller fragments may yet contain the regulatory properties of thepromoter so identified and deletion analysis is one method ofidentifying essential regions. Deletion analysis can occur from both the5′ and 3′ ends of the regulatory region. Fragments can be obtained bysite-directed mutagenesis, mutagenesis using the polymerase chainreaction and the like. (See, Directed Mutagenesis: A Practical ApproachIRL Press (1991)). The 3′ deletions can delineate the essential regionand identify the 3′ end so that this region may then be operably linkedto a core promoter of choice. Once the essential region is identified,transcription of an exogenous gene may be controlled by the essentialregion plus a core promoter. By core promoter is meant the sequencecalled the TATA box which is common to promoters in all genes encodingproteins. Thus the upstream promoter of Ms26 can optionally be used inconjunction with its own or core promoters from other sources. Thepromoter may be native or non-native to the cell in which it is found.

The core promoter can be any one of known core promoters such as theCauliflower Mosaic Virus 35S or 19S promoter (U.S. Pat. No. 5,352,605),ubiquitin promoter (U.S. Pat. No. 5,510,474) the 1N2 core promoter (U.S.Pat. No. 5,364,780) or a Figwort Mosaic Virus promoter (Gruber, et al.“Vectors for Plant Transformation” Methods in Plant Molecular Biologyand Biotechnology) et al. eds, CRC Press pp. 89-119 (1993)).

The regulatory region of Ms26 has been identified as including the 1005bp region upstream of the putative TATA box. See FIG. 8. Further, usingthe procedures outlined above, it has been determined that an essentialregion of the promoter includes the −180 bp upstream of the TATA box andspecifically, the −176 to −44 region is particularly essential.

Promoter sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the promotersequence set forth herein. In these techniques, all or part of the knownpromoter sequence is used as a probe which selectively hybridizes toother sequences present in a population of cloned genomic DNA fragments(i.e. genomic libraries) from a chosen organism. Methods are readilyavailable in the art for the hybridization of nucleic acid sequences.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g. Innis et al., eds., (1990) PCR Protocols,A Guide to Methods and Applications, Academic Press).

Further, a promoter of the present invention can be linked withnucleotide sequences other than the Ms26 gene to express otherheterologous nucleotide sequences. The nucleotide sequence for thepromoter of the invention, as well as fragments and variants thereof,can be provided in expression cassettes along with heterologousnucleotide sequences for expression in the plant of interest, moreparticularly in the male tissue of the plant. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the nucleotide sequence to be under the transcriptional regulation ofthe promoter. These expression cassettes are useful in the geneticmanipulation of any plant to achieve a desired phenotypic response.

Examples of other nucleotide sequences which can be used as theexogenous gene of the expression vector with the Ms26 promoter, or otherpromoters taught herein or known to those of skill in the art, or otherpromoters taught herein or known to those of skill in the artcomplementary nucleotidic units such as antisense molecules (callaseantisense RNA, barnase antisense RNA and chalcone synthase antisenseRNA, Ms45 antisense RNA), ribozymes and external guide sequences, anaptamer or single stranded nucleotides. The exogenous nucleotidesequence can also encode carbohydrate degrading or modifying enzymes,amylases, debranching enzymes and pectinases, such as the alpha amylasegene of FIG. 24, auxins, rol B, cytotoxins, diptheria toxin, DAMmethylase, avidin, or may be selected from a prokaryotic regulatorysystem. By way of example, Mariani, et al., Nature Vol. 347; pp. 737;(1990), have shown that expression in the tapetum of either Aspergillusoryzae RNase-T1 or an RNase of Bacillus amyloliquefaciens, designated“barnase,” induced destruction of the tapetal cells, resulting in maleinfertility. Quaas, et al., Eur. J. Biochem. Vol. 173: pp. 617 (1988),describe the chemical synthesis of the RNase-T1, while the nucleotidesequence of the barnase gene is disclosed in Hartley, J. Molec. Biol.;Vol. 202: pp. 913 (1988). The rolB gene of Agrobacterium rhizogenescodes for an enzyme that interferes with auxin metabolism by catalyzingthe release of free indoles from indoxyl-β-glucosides. Estruch, et al.,EMBO J. Vol. 11: pp. 3125 (1991) and Spena, et al., Theor. Appl. Genet.;Vol. 84: pp. 520 (1992), have shown that the anther-specific expressionof the rolB gene in tobacco resulted in plants having shriveled anthersin which pollen production was severely decreased and the rolB gene isan example of a gene that is useful for the control of pollenproduction. Slightom, et al., J. Biol. Chem. Vol. 261: pp. 108 (1985),disclose the nucleotide sequence of the rolB gene. DNA moleculesencoding the diphtheria toxin gene can be obtained from the AmericanType Culture Collection (Rockville, Md.), ATCC No. 39359 or ATCC No.67011 and see Fabijanski, et al., E.P. Appl. No. 90902754.2, “MolecularMethods of Hybrid Seed Production” for examples and methods of use. TheDAM methylase gene is used to cause sterility in the methods discussedat U.S. Pat. No. 5,689,049 and PCT/US95/15229 Cigan, A. M. andAlbertsen, M. C., “Reversible Nuclear Genetic System for Male Sterilityin Transgenic Plants.” Also see discussion of use of the avidin gene tocause sterility at U.S. Pat. No. 5,962,769 “Induction of Male Sterilityin Plants by Expression of High Levels of Avidin” by Albertsen et al.

The invention includes vectors with the Ms26 gene. A vector is preparedcomprising Ms26, a promoter that will drive expression of the gene inthe plant and a terminator region. As noted, the promoter in theconstruct may be the native promoter or a substituted promoter whichwill provide expression in the plant. The promoter in the construct maybe an inducible promoter, so that expression of the sense or antisensemolecule in the construct can be controlled by exposure to the inducer.In this regard, any plant-compatible promoter elements can be employedin the construct, influenced by the end result desired. Those can beplant gene promoters, such as, for example, the promoter for the smallsubunit of ribulose-1,5-bis-phosphate carboxylase, or promoters from thetumor-inducing plasmids from Agrobacterium tumefaciens, such as thenopaline synthase and octopine synthase promoters, or viral promoterssuch as the cauliflower mosaic virus (CaMV) 19S and 35S promoters or thefigwort mosaic virus 35S promoter. See Kay et al., (1987) Science236:1299 and European patent application No. 0 342 926; the barley lipidtransfer protein promoter, LTP2 (Kalla et al., Plant J. (1994) 6(6):849-60); the ubiquitin promoter (see for example U.S. Pat. No.5,510,474); the END2 promoter (Linnestad et al. U.S. Pat. No.6,903,205); and the polygalacturonase PG47 promoter (See Allen andLonsdale, Plant J. (1993) 3:261-271; WO 94/01572; U.S. Pat. No.5,412,085). See international application WO 91/19806 for a review ofillustrative plant promoters suitably employed in the present invention.

The range of available plant compatible promoters includes tissuespecific and inducible promoters. An inducible regulatory element is onethat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer the DNA sequences or genes will not betranscribed. Typically the protein factor that binds specifically to aninducible regulatory element to activate transcription is present in aninactive form which is then directly or indirectly converted to theactive form by the inducer. The inducer can be a chemical agent such asa protein, metabolite, growth regulator, herbicide or phenolic compoundor a physiological stress imposed directly by heat, cold, salt, or toxicelements or indirectly through the actin of a pathogen or disease agentsuch as a virus. A plant cell containing an inducible regulatory elementmay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol. 22: 361-366 (1993). Exemplary inducible promotersinclude ecdysone receptor promoters, U.S. Pat. No. 6,504,082; promotersfrom the ACE1 system which responds to copper (Mett et al. PNAS 90:4567-4571 (1993)); In2-1 and In2-2 gene from maize which respond tobenzenesulfonamide herbicide safeners (U.S. Pat. No. 5,364,780; Hersheyet al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al., Mol.Gen. Genetics 243: 32-38 (1994)); the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides; and the tobacco PR-1a promoter, which isactivated by salicylic acid. Other chemical-regulated promoters ofinterest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.14(2):247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet.227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhancedtranscription and/or expression within a particular plant tissue.Promoters may express in the tissue of interest, along with expressionin other plant tissue, may express strongly in the tissue of interestand to a much lesser degree than other tissue, or may express highlypreferably in the tissue of interest. Tissue-preferred promoters includethose described in Yamamoto et al. (1997) Plant J. 12(2): 255-265;Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al.(1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) TransgenicRes. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535;Canevascini et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto etal. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl.Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3): 495-505. Inone embodiment, the promoters are those which preferentially express tothe male or female tissue of the plant. The invention does not requirethat any particular male tissue-preferred promoter be used in theprocess, and any of the many such promoters known to one skilled in theart may be employed. The native Ms26 promoter described herein is oneexample of a useful promoter. Another such promoter is the 5126promoter, which preferentially directs expression of the gene to whichit is linked to male tissue of the plants, as described in U.S. Pat.Nos. 5,837,851 and 5,689,051. Other examples include the Ms45 promoterdescribed at U.S. Pat. No. 6,037,523; SF3 promoter described at U.S.Pat. No. 6,452,069; the BS92-7 promoter described at WO 02/063021; aSGB6 regulatory element described at U.S. Pat. No. 5,470,359; the TA29promoter (Koltunow et al. (1990) “Different temporal and spatial geneexpression patterns occur during anther development.” Plant Cell2:1201-1224; Goldberg, R. B., Beals, T. P. and Sanders, P. M., (1993)“Anther development: basic principles and practical applications” PlantCell 5:1217-1229; and U.S. Pat. No. 6,399,856); the type 2metallothionein-like gene promoter (Charbonnel-Campaa et al., Gene(2000) 254:199-208); and the Brassica Bca9 promoter (Lee et al., PlantCell Rep. (2003) 22:268-273).

Male gamete preferred promoters include the PG47 promoter, supra as wellas ZM13 promoter (Hamilton et al., Plant Mol. Biol. (1998) 38:663-669);actin depolymerizing factor promoters (such as Zmabp1, Zmabp2; see forexample Lopez et al. Proc. Natl. Acad. Sci. USA (1996) 93: 7415-7420);the promoter of the maize petctin methylesterase-liked gene, ZmC5(Wakeley et al. Plant Mol. Biol. (1998) 37:187-192); the profiling genepromoter Zmpro1 (Kovar et al., The Plant Cell (2000) 12:583-598); thesulphated pentapeptide phytosulphokine gene ZmPSK1 (Lorbiecke et al.,Journal of Experimental Botany (2005) 56(417): 1805-1819); the promoterof the calmodulin binding protein Mpcbp (Reddy et al. J. Biol. Chem.(2000) 275(45):35457-70).

Other components of the vector may be included, also depending uponintended use of the gene. Examples include selectable markers, targetingor regulatory sequences, stabilizing or leader sequences, introns etc.General descriptions and examples of plant expression vectors andreporter genes can be found in Gruber, et al., “Vectors for PlantTransformation” in Method in Plant Molecular Biology and Biotechnology,Glick et al eds;CRC Press pp. 89-119 (1993). The selection of anappropriate expression vector will depend upon the host and the methodof introducing the expression vector into the host. The expressioncassette will also include at the 3′ terminus of the heterologousnucleotide sequence of interest, a transcriptional and translationaltermination region functional in plants. The termination region can benative with the promoter nucleotide sequence of the present invention,can be native with the DNA sequence of interest, or can be derived fromanother source. Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also, Guerineau et al. Mol. Gen.Genet. 262:141-144 (1991); Proudfoot, Cell 64:671-674 (1991); Sanfaconet al. Genes Dev. 5:141-149 (1991); Mogen et al. Plant Cell 2:1261-1272(1990); Munroe et al. Gene 91:151-158 (1990); Ballas et al. NucleicAcids Res. 17:7891-7903 (1989); Joshi et al. Nucleic Acid Res.15:9627-9639 (1987).

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include by way of example, picornavirusleaders, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. Proc. Nat. Acad. Sci. USA 86:6126-6130 (1989);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allisonet al.; MDMV leader (Maize Dwarf Mosaic Virus), Virology 154:9-20(1986); human immunoglobulin heavy-chain binding protein (BiP), Macejaket al. Nature 353:90-94 (1991); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. Nature325:622-625 (1987); Tobacco mosaic virus leader (TMV), Gallie et al.(1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV) Lommel et al. Virology 81:382-385 (1991). Seealso Della-Cioppa et al. Plant Physiology 84:965-968 (1987). Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence directed to a particularorganelle, particularly the plastid, amyloplast, or to the endoplasmicreticulum, or secreted at the cell's surface or extracellularly, theexpression cassette can further comprise a coding sequence for a transitpeptide. Such transit peptides are well known in the art and include,but are not limited to, the transit peptide for the acyl carrierprotein, the small subunit of RUBISCO, plant EPSP synthase, Zea maysBrittle-1 chloroplast transit peptide (Nelson et al. Plant physiol117(4):1235-1252 (1998); Sullivan et al. Plant Cell 3(12):1337-48;Sullivan et al., Planta (1995) 196(3):477-84; Sullivan et al., J. Biol.Chem. (1992) 267(26):18999-9004) and the like. One skilled in the artwill readily appreciate the many options available in expressing aproduct to a particular organelle. For example, the barley alpha amylasesequence is often used to direct expression to the endoplasmic reticulum(Rogers, J. Biol. Chem. 260:3731-3738 (1985)). Use of transit peptidesis well known (e.g., see U.S. Pat. Nos. 5,717,084; 5,728,925).

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions, such astransitions and transversions, can be involved.

As noted herein, the present invention provides vectors capable ofexpressing genes of interest. In general, the vectors should befunctional in plant cells. At times, it may be preferable to havevectors that are functional in E. coli (e.g., production of protein forraising antibodies, DNA sequence analysis, construction of inserts,obtaining quantities of nucleic acids). Vectors and procedures forcloning and expression in E. coli are discussed in Sambrook et al.(supra).

The transformation vector comprising the promoter sequence of thepresent invention operably linked to a heterologous nucleotide sequencein an expression cassette, can also contain at least one additionalnucleotide sequence for a gene to be cotransformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. Mol.Cell. Biol. 7:725-737 (1987); Goff et al. EMBO J. 9:2517-2522 (1990);Kain et al. BioTechniques 19:650-655 (1995); and Chiu et al. CurrentBiology 6:325-330 (1996).

Selectable reporter genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to,genes encoding resistance to chloramphenicol, Herrera Estrella et al.EMBO J. 2:987-992 (1983); methotrexate, Herrera Estrella et al. Nature303:209-213 (1983); Meijer et al. Plant Mol. Biol. 16:807-820 (1991);hygromycin, Waldron et al. Plant Mol. Biol. 5:103-108 (1985), Zhijian etal. Plant Science 108:219-227 (1995); streptomycin, Jones et al. Mol.Gen. Genet. 210:86-91 (1987); spectinomycin, Bretagne-Sagnard et al.Transgenic Res. 5:131-137 (1996); bleomycin, Hille et al. Plant Mol.Biol. 7:171-176 (1990); sulfonamide, Guerineau et al. Plant Mol. Biol.15:127-136 (1990); bromoxynil, Stalker et al. Science 242:419-423(1988); glyphosate, Shaw et al. Science 233:478-481 (1986); andphosphinothricin, DeBlock et al. EMBO J. 6:2513-2518 (1987).

Scorable or screenable markers may also be employed, where presence ofthe sequence produces a measurable product. Examples include aβ-glucuronidase, or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known (for example, U.S. Pat. Nos.5,268,463 and 5,599,670); chloramphenicol acetyl transferase (Jeffersonet al. The EMBO Journal vol. 6 No. 13 pp. 3901-3907); and alkalinephosphatase. Other screenable markers include the anthocyanin/flavonoidgenes in general (See discussion at Taylor and Briggs, The Plant Cell(1990) 2:115-127) including, for example, a R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., in Chromosome Structure andFunction, Kluwer Academic Publishers, Appels and Gustafson eds., pp.263-282 (1988)); the genes which control biosynthesis of flavonoidpigments, such as the maize C1 gene (Kao et al., Plant Cell (1996) δ:1171-1179; Scheffler et al. Mol. Gen. Genet. (1994) 242:40-48) and maizeC2 (Wienand et al., Mol. Gen. Genet. (1986) 203:202-207); the B gene(Chandler et al., Plant Cell (1989) 1:1175-1183), the p1 gene (Grotewoldet al, Proc. Natl. Acad. Sci. USA (1991) 88:4587-4591; Grotewold et al.,Cell (1994) 76:543-553; Sidorenko et al., Plant Mol. Biol. (1999)39:11-19); the bronze locus genes (Ralston et al., Genetics (1988)119:185-197; Nash et al., Plant Cell (1990) 2(11): 1039-1049), amongothers. Yet further examples of suitable markers include the cyanfluorescent protein (CYP) gene (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129: 913-42), the yellowfluorescent protein gene (PhiYFP™ from Evrogen; see Bolte et al. (2004)J. Cell Science 117: 943-54); a lux gene, which encodes a luciferase,the presence of which may be detected using, for example, X-ray film,scintillation counting, fluorescent spectrophotometry, low-light videocameras, photon counting cameras or multiwell luminometry (Teeri et al.(1989) EMBO J. 8:343); a green fluorescent protein (GFP) gene (Sheen etal., Plant J. (1995) 8(5):777-84); and DsRed2 where plant cellstransformed with the marker gene are red in color, and thus visuallyselectable (Dietrich et al. (2002) Biotechniques 2(2):286-293).Additional examples include a p-lactamase gene (Sutcliffe, Proc. Nat'l.Acad. Sci. U.S.A. (1978) 75:3737), which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l. Acad. Sci.U.S.A. (1983) 80:1101), which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta et al., Biotech.(1990) 8:241); and a tyrosinase gene (Katz et al., J. Gen. Microbiol.(1983) 129:2703), which encodes an enzyme capable of oxidizing tyrosineto DOPA and dopaquinone, which in turn condenses to form the easilydetectable compound melanin. Clearly, many such markers are available toone skilled in the art.

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription or transcript andtranslation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for efficienttransformation/transfection may be employed.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. See, forexample, Miki et al, “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biotechnology, supra; Klein et al,Bio/Technology 10:268 (1992); and Weising et al., Ann. Rev. Genet. 22:421-477 (1988). For example, the DNA construct may be introduced intothe genomic DNA of the plant cell using techniques such asmicroprojectile-mediated delivery, Klein et al., Nature 327: 70-73(1987); electroporation, Fromm et al., Proc. Natl. Acad. Sci. 82: 5824(1985); polyethylene glycol (PEG) precipitation, Paszkowski et al., EMBOJ. 3: 2717-2722 (1984); direct gene transfer WO 85/01856 and EP No. 0275 069; in vitro protoplast transformation, U.S. Pat. No. 4,684,611;and microinjection of plant cell protoplasts or embryogenic callus,Crossway, Mol. Gen. Genetics 202:179-185 (1985). Co-cultivation of planttissue with Agrobacterium tumefaciens is another option, where the DNAconstructs are placed into a binary vector system. See e.g., U.S. Pat.No. 5,591,616; Ishida et al., “High Efficiency Transformation of Maize(Zea mays L.) mediated by Agrobacterium tumefaciens” NatureBiotechnology 14:745-750 (1996). The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct into the plant cell DNA when the cell is infected by thebacteria. See, for example Horsch et al., Science 233: 496-498 (1984),and Fraley et al., Proc. Natl. Acad. Sci. 80: 4803 (1983).

Standard methods for transformation of canola are described at Moloneyet al. “High Efficiency Transformation of Brassica napus usingAgrobacterium Vectors” Plant Cell Reports 8:238-242 (1989). Corntransformation is described by Fromm et al, Bio/Technology 8:833 (1990)and Gordon-Kamm et al, supra. Agrobacterium is primarily used in dicots,but certain monocots such as maize can be transformed by Agrobacterium.See supra and U.S. Pat. No. 5,550,318. Rice transformation is describedby Hiei et al., “Efficient Transformation of Rice (Oryza sativs L.)Mediated by Agrobacterium and Sequence Analysis of the Boundaries of theT-DNA” The Plant Journal 6(2): 271-282 (1994, Christou et al, Trends inBiotechnology 10:239 (1992) and Lee et al, Proc. Nat'l Acad. Sci. USA88:6389 (1991). Wheat can be transformed by techniques similar to thoseused for transforming corn or rice. Sorghum transformation is describedat Casas et al, supra and sorghum by Wan et al, Plant Physicol. 104:37(1994). Soybean transformation is described in a number of publications,including U.S. Pat. No. 5,015,580.

When referring to “introduction” of the nucleotide sequence into aplant, it is meant that this can occur by direct transformation methods,such as Agrobacterium transformation of plant tissue, microprojectilebombardment, electroporation, or any one of many methods known to oneskilled in the art; or, it can occur by crossing a plant having theheterologous nucleotide sequence with another plant so that progeny havethe nucleotide sequence incorporated into their genomes. Such breedingtechniques are well known to one skilled in the art.

The plant breeding methods used herein are well known to one skilled inthe art. For a discussion of plant breeding techniques, see Poehlman(1987) Breeding Field Crops. AVI Publication Co., Westport Conn. Many ofthe plants which would be most preferred in this method are bred throughtechniques that take advantage of the plant's method of pollination.

Backcrossing methods may be used to introduce a gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPlant Breeding Methodology, edit. Neal Jensen, John Wiley & Sons, Inc.(1988). In a typical backcross protocol, the original variety ofinterest (recurrent parent) is crossed to a second variety (nonrecurrentparent) that carries the single gene of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a plant is obtainedwherein essentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

In certain embodiments of the invention, it is desirable to maintain themale sterile homozygous recessive condition of a male sterile plant,when using a transgenic restoration approach, while decreasing thenumber of plants, plantings and steps needed for maintenance plant withsuch traits. Homozygosity is a genetic condition existing when identicalalleles reside at corresponding loci on homologous chromosomes.Heterozygosity is a genetic condition existing when different allelesreside at corresponding loci on homologous chromosomes. Hemizygosity isa genetic condition existing when there is only one copy of a gene (orset of genes) with no allelic counterpart on the sister chromosome. Inan embodiment, the homozygous recessive condition results in conferringon the plant a trait of interest, which can be any trait desired andwhich results from the recessive genotype, such as increased drought orcold tolerance, early maturity, changed oil or protein content, or anyof a multitude of the many traits of interest to plant breeders. In oneembodiment, the homozygous recessive condition confers male sterilityupon the plant. When the sequence which is the functional complement ofthe homozygous condition is introduced into the plant (that is, asequence which, when introduced into and expressed in the plant havingthe homozygous recessive condition, restores the wild-type condition),fertility is restored by virtue of restoration of the wild-type fertilephenotype.

Maintenance of the homozygous recessive condition is achieved byintroducing a restoration transgene construct into a plant that islinked to a sequence which interferes with the function or formation ofmale gametes of the plant to create a maintainer or donor plant. Therestoring transgene, upon introduction into a plant that is homozygousrecessive for the genetic trait, restores the genetic function of thattrait, with the plant producing only viable pollen containing a copy ofthe recessive allele but does not contain the restoration transgene. Thetransgene is kept in the hemizygous state in the maintainer plant. Bytransgene, it is meant any nucleic acid sequence which is introducedinto the genome of a cell by genetic engineering techniques. A transgenemay be a native DNA sequence, or a heterologous DNA sequence (i.e.,“foreign DNA”). The term native DNA sequence refers to a nucleotidesequence which is naturally found in the cell but that may have beenmodified from its original form. The pollen from the maintainer can beused to fertilize plants that are homozygous for the recessive trait,and the progeny will therefore retain their homozygous recessivecondition. The maintainer plant containing the restoring transgeneconstruct is propagated by self-fertilization, with the resulting seedused to produce further plants that are homozygous recessive plants andcontain the restoring transgene construct.

The maintainer plant serves as a pollen donor to the plant having thehomozygous recessive trait. The maintainer is optimally produced from aplant having the homozygous recessive trait and which also hasnucleotide sequences introduced therein which would restore the traitcreated by the homozygous recessive alleles. Further, the restorationsequence is linked to nucleotide sequences which interfere with thefunction or formation of male gametes. The gene can operate to preventformation of male gametes or prevent function of the male gametes by anyof a variety of well-know modalities and is not limited to a particularmethodology. By way of example but not limitation, this can include useof genes which express a product cytotoxic to male gametes (See forexample, U.S. Pat. Nos. 5,792,853; 5,689,049; PCT/EP89/00495); inhibitproduct formation of another gene important to male gamete function orformation (See, U.S. Pat. Nos. 5,859,341; 6,297,426); combine withanother gene product to produce a substance preventing gene formation orfunction (See U.S. Pat. Nos. 6,162,964; 6,013,859; 6,281,348; 6,399,856;6,248,935; 6,750,868; 5,792,853); are antisense to or causeco-suppression of a gene critical to male gamete function or formation(See U.S. Pat. Nos. 6,184,439; 5,728,926; 6,191,343; 5,728,558;5,741,684); interfere with expression through use of hairpin formations(Smith et al. (2000) Nature 407:319-320; WO 99/53050 and WO 98/53083) orthe like. Many nucleotide sequences are known which inhibit pollenformation or function and any sequences which accomplish this functionwill suffice. A discussion of genes which can impact proper developmentor function is included at U.S. Pat. No. 6,399,856 and includes dominantnegative genes such as cytotoxin genes, methylase genes, andgrowth-inhibiting genes. Dominant negative genes include diphtheriatoxin A-chain gene (Czako, M. and An, G. (1991) “Expression of DNAcoding for Diptheria toxin Chain A is toxic to plant cells” PlantPhysiol. 95 687-692. and Greenfield et al PNAS 80:6853 (1983), Palmiteret al Cell 50:435 (1987)); cell cycle division mutants such as CDC inmaize (Colasanti, J., Tyers, M. and Sundaresan, V., “Isolation andCharacterization of cDNA clones encoding a functional P34 cdc2 homologuefrom Zea mays” PNAS 88, 3377-3381 (1991)); the WT gene (Farmer, A. A.,Loftus, T. M., Mills, A. A., Sato, K. V., Neill, J., Yang, M., Tron, T.,Trumpower, B. L. and Stanbridge, E. G. Hum. Mol. Genet. 3, 723-728(1994)); and P68 (Chen, J. J., Pal, J. K., Petryshyn, R., Kuo, I., Yang,J. M., Throop, M. S., Gehrke, L. and London, I. M. “Eukaryotictranslation initiation kinases” PNAS 88, 315-319 (1991)).

Further examples of so-called “cytotoxic” genes are discussed supra andcan include, but are not limited to pectate lyase gene pelE, fromErwinia chrysanthermi (Kenn et al J. Bacteroil 168:595 (1986)); T-urf13gene from cms-T maize mitochondrial genomes (Braun et al Plant Cell2:153 (1990); Dewey et al. PNAS 84:5374 (1987)); CytA toxin gene fromBacillus thuringiensis Israeliensis that causes cell membrane disruption(McLean et al J. Bacteriol 169:1017 (1987), U.S. Pat. No. 4,918,006);DNAses, RNAses, (U.S. Pat. No. 5,633,441); proteases, or a genesexpressing anti-sense RNA. A suitable gene may also encode a proteininvolved in inhibiting pistil development, pollen stigma interactions,pollen tube growth or fertilization, or a combination thereof. Inaddition genes that either interfere with the normal accumulation ofstarch in pollen or affect osmotic balance within pollen may also besuitable.

In an illustrative embodiment, the DAM-methylase gene is used, discussedsupra and at U.S. Pat. Nos. 5,792,852 and 5,689,049, the expressionproduct of which catalyzes methylation of adenine residues in the DNA ofthe plant. Methylated adenines will affect cell viability and will befound only in the tissues in which the DAM-methylase gene is expressed.In another embodiment, an α-amylase gene can be used with a maletissue-preferred promoter. During the initial germinating period ofcereal seeds, the aleurone layer cells will synthesize α-amylase, whichparticipates in hydrolyzing starch to form glucose and maltose, so as toprovide the nutrients needed for the growth of the germ (J. C. Rogersand C. Milliman, J. Biol. Chem., 259 (19): 12234-12240, 1984; Rogers, J.C., J. Biol. Chem., 260: 3731-3738, 1985). In an embodiment, theα-amylase gene used can be the Zea mays α-amylase-1 gene. Young et al.“Cloning of an α-amylase cDNA from aleurone tissue of germinating maizeseed” Plant Physiol. 105(2) 759-760 and GenBank accession No. L25805,GI:426481). Sequences encoding α-amylase are not typically found inpollen cells, and when expression is directed to male tissue, the resultis a breakdown of the energy source for the pollen grains, andrepression of pollen development.

One skilled in this area readily appreciates the methods describedherein are applicable to any other crops which have the potential tooutcross. By way of example, but not limitation it can include maize,soybean, sorghum, or any plant with the capacity to outcross.

Ordinarily, to produce more plants having the recessive condition, onemight cross the recessive plant with another recessive plant. This maynot be desirable for some recessive traits and may be impossible forrecessive traits affecting reproductive development. Alternatively, onecould cross the homozygous plant with a second plant having therestoration gene, but this requires further crossing to segregate awaythe restoring gene to once again reach the recessive phenotypic state.Instead, in one process the homozygous recessive condition can bemaintained, while crossing it with the maintainer plant. This method canbe used with any situation in which is it desired to continue therecessive condition. This results in a cost-effective system that isrelatively easy to operate to maintain a population of homozygousrecessive plants.

A sporophytic gene is one which operates independently of the gametes.When the homozygous recessive condition is one which produces malesterility by preventing male sporophyte development, the maintainerplant, of necessity, must contain a functional restoring transgeneconstruct capable of complementing the mutation and rendering thehomozygous recessive plant able to produce viable pollen. Linking thissporophytic restoration gene with a second functional nucleotidesequence which interferes with the function or formation of the malegametes of the plant results in a maintainer plant that produces pollencontaining only the recessive allele of the sporophytic gene at the itsnative locus due to the action of the second nucleotide sequence ininterfering with pollen formation or function. This viable pollenfraction is non-transgenic with regard to the restoring transgeneconstruct.

In a still further embodiment, a marker gene, as discussed supra, may beprovided in the construct with the restoring transgene. By way ofexample without limitation, use of a herbicide resistant marker, such asbar allows one to eliminate cells not having the restoring transgene. Inyet another example, when using a scorable marker, such as a redfluorescent marker, such as DsRed2, any inadvertent transmission of thetransgene can also be detected visually, and such escapes eliminatedfrom progeny. Clearly, many other variations in the restoring constructare available to one skilled in the art.

In an illustrative embodiment, a method of maintaining a homozygousrecessive condition of a male sterile plant at a genetic locus isprovided, in which is employed a first nucleotide sequence which is agene critical to male fertility, a second nucleotide sequence whichinhibits the function or formation of viable male gametes, an optionalthird nucleotide sequence which is operably linked to the first sequenceand preferentially expresses the sequence in male plant cells, anoptional fourth nucleotide sequence operably linked to a fourthnucleotide sequence, the fourth sequence directing expression to malegametes, and an optional fifth nucleotide sequence which is a selectableor scorable marker allowing for selection of plant cells.

For example, it is desirable to produce male sterile female plants foruse in the hybrid production process which are sterile as a result ofbeing homozygous for a mutation in the Ms45 gene; a gene, which iscritical to male fertility. Such a mutant Ms45 allele is designated asms45 and a plant that is homozygous for ms45 (represented by thenotation ms45/ms45) displays the homozygous recessive male sterilityphenotype and produces no functional pollen. See, U.S. Pat. Nos.5,478,369; 5,850,014; 6,265,640; and 5,824,524. In both the inbred andhybrid production processes, it is highly desired to maintain thishomozygous recessive condition. When sequences encoding the Ms45 geneare introduced into a plant having the homozygous condition, malefertility results. By the method of the invention, a plant which isms45/ms45 homozygous recessive may have introduced into it a functionalsporophytic Ms45 gene, and thus is male fertile. This gene can be linkedto a gene which operates to render pollen containing the restoringtransgene construct nonfunctional or prevents its formation, or whichproduces a lethal product in pollen, linked to the promoter directingits expression to the male gametes to produce a plant that only producedpollen containing ms45 without the restoring transgene construct.

An example is a construct which includes the Ms45 gene, linked with a5126 promoter, a male tissue-preferred promoter (See U.S. Pat. Nos.5,750,868; 5,837,851; and 5,689,051) and further linked to the cytotoxicDAM methylase gene under control of the polygalacturonase promoter, PG47promoter (See U.S. Pat. Nos. 5,792,853; 5,689,049) in a hemizygoticcondition. Therefore the resulting plant produces pollen, but the onlyviable pollen results from the alle not containing the resoring Ms45/DAMmethylase construct and thus contains only the ms45 gene. It cantherefore be used as a pollinator to fertilize the homozygous recessiveplant (ms45/ms45), and progeny produced will continue to be male sterileas a result of maintaining homozygosity for ms45. The progeny will alsonot contain the introduced restoring transgene construct.

In yet another restoring construct example, the Ms26 gene is linked witha 5126 promoter, and further linked to the Zea mays α-amylase gene undercontrol of the male tissue-preferred PG47 promoter. The scorable markerused in an embodiment is DS-RED EXPRESS.

A desirable result of the process of the invention is that the planthaving the restorer nucleotide sequence may be self-fertilized, that ispollen from the plant transferred to the flower of the same plant toachieve the propagation of restorer plants. (Note that in referring to“self fertilization”, it includes the situation where the plantproducing the pollen is fertilized with that same the pollen, and thesituation where two or more identical inbred plants are planted togetherand pollen from the identical inbred plant pollinate a differentidentical inbred plant). The pollen will not have the restoringtransgene construct but it will be contained in 50% of the ovules (thefemale gamete). The seed resulting from the self-fertilization can beplanted, and selection made for the seed having the restoring transgeneconstruct. The selection process can occur by any one of many knownprocesses; the most common where the restoration nucleotide sequence islinked to a marker gene. The marker can be scorable or selectable, andallows those plants produced from the seed having the restoration geneto be identified.

In an embodiment of the invention, it is possible to provide that themale gamete-tissue preferred promoter is inducible. Additional controlis thus allowed in the process, where so desired, by providing that theplant having the restoration nucleotide sequences is constitutively malesterile. This type of male sterility is set forth the in U.S. Pat. No.5,859,341. In order for the plant to become fertile, the inducingsubstance must be provided, and the plant will become fertile. Again,when combined with the process of the invention as described supra, theonly pollen produced will not contain the restoration nucleotidesequences.

Further detailed description is provided below by way of instruction andillustration and is not intended to limit the scope of the invention.

Example 1 Identification and Cosegregation of ms26-m2::Mu8

Families of plants from a Mutator (Mu) population were identified thatsegregated for plants that were mostly male sterile, with none or only afew extruded abnormal anthers, none of which had pollen present. Malesterility is expected to result from those instances where a Mu elementhas randomly integrated into a gene responsible for some step inmicrosporogenesis, disrupting its expression. Plants from a segregatingF₂ family in which the male sterile mutation was designatedms26*-SBMu200, were grown and classified for male fertility/sterilitybased on the above criteria. Leaf samples were taken and DNAsubsequently isolated on approximately 20 plants per phenotypicclassification, that is male fertility vs. male sterility.

Southern analysis was performed to confirm association of Mu withsterility. Southern analysis is a well known technique to those skilledin the art. This common procedure involves isolating the plant DNA,cutting with restriction endonucleases, fractioning the cut DNA bymolecular weight on an agarose gel, and transferring to nylon membranesto fix the separated DNA. These membranes are subsequently hybridizedwith a probe fragment that was radioactively labeled with P³²P-dCTP, andwashed in an SDS solution. Southern, E., “Detection of SpecificSequences Among DNA Fragments by Gel Electrophoresis,” J. Mol. Biol.98:503-317 (1975). Plants from a segregating F₂ ms26*-SBMu200 familywere grown and classified for male fertility/sterility. Leaf samples andsubsequent DNA isolation was conducted on approximately 20 plants perphenotypic classification. DNA (˜7 ug) from 5 fertile and 12 sterileplants was digested with EcoRI and electrophoresed through a 0.75%agarose gel. The digested DNA was transferred to nylon membrane viaSouthern transfer. The membrane was hybridized with an internal fragmentfrom the Mu8 transposon. Autoradiography of the membrane revealedcosegregation of an approximately 5.6 Kb EcoRI fragment with thesterility phenotype as shown in FIG. 1. This EcoRI band segregated inthe fertile plants suggesting a heterozygous wild type condition for theallele

Example 2 Library Construction, Screening, and Mapping

The process of genomic library screenings is commonly known among thoseskilled in the art and is described at Sambrook, J., Fritsch, E. F.,Maniatis T., et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor Lab Press, Plainview, N.Y. (1989).Libraries were created as follows.

DNA from a sterile plant was digested with EcoRI and run on apreparative gel. DNA with a molecular weight between 5.0 and 6.0 Kb wasexcised from the gel, electroeluted and ethanol precipitated. This DNAwas ligated into the Lambda Zap vector (Stratagene™) using themanufacturer's protocol. The ligated DNA was packaged into phageparticles using Gigapack Gold (Stratagene™). Approximately 500,000 PFUwere plated and lifted onto nitrocellulose membranes. Membranes werehybridized with the Mu8 probe. A pure clone was obtained after 3 roundsof screening. The insert was excised from the phage as a plasmid anddesignated SBMu200-3.1. A PstI border fragment from this clone wasisolated and used to reprobe the orginal EcoRI cosegregation blot asshown in FIG. 2B. The approximately 5.6 kb EcoRI fragment is homozygousin all the sterile plants, which confirms that the correct Mu fragmentwas isolated. Three of the fertile plants are heterozygous for the 5.5kb EcoRI band and a 4.3 Kb EcoRI band. Two of the fertile plants arehomozygous for the 4.3 kb EcoRI band, presumably the wild type allele.

The PstI probe was used to map the ms*-SBMu200 mutation in an RFLPmapping population. The mutant mapped to the short arm of chromosome 1,near the male sterile locus, Ms26 (Loukides et al., (1995) Amer. J. Bot82, 1017-1023). To test whether ms*-SBMu200 was an allele of ms26-ref,ms*-SBMu200 and ms26-ref were crossed with each other using a knownheterozygote as the pollen donor. The testcross progeny segregatedmale-sterile and wild-type plants in a 1:1 ratio, indicating allelismbetween ms*-SBMu200 and ms26-ref. The ms*-SBMu200 allele was designatedms26-m2::Mu8. The map location is shown in FIG. 13.

Example 3 Identification and Cloning of Additional ms26 Alleles

An additional Mu insertion mutations in Ms26 was identified by using apolymerase chain reaction (PCR) primer for Mu and a gene specific primerfor Ms26 and screening a population of Mu F₁ families. Sequence analysesof the PCR products showed that all three Mu insertions occurred in thesecond exon (FIG. 1). The F₂ seeds from one of these families were grownand examined for male fertility/sterility. Southern blot analyses ofthis family confirmed the cosegregation of the Mu insertion in Ms26 withthe male-sterile phenotype and the allele was designated ms26-m3:: Mu.

The ms26 allele described in Loukides et al., (1995) Amer. J. Bot 82,1017-1023 and designated ms26-ref was also investigated. To analyze themutation in ms26-ref, Ms26 genomic sequences were cloned from ms26-refsterile and fertile plants. Ms26 was cloned as a ˜4.2 kb EcoRI fragmentand ms26-ref cloned as a ˜6 kb HindII fragment and an overlapping ˜2.3kb EcoRI fragment from the sterile plant. Sequence analysis revealed thepresence of a new segment (1,430 bp) in the last exon of the ms26-refallele shown in FIG. 1. An 8 by host site duplication (GCCGGAGC) wasfound that flanks the inserted element and the element also contains a15 bp terminal inverted repeat (TIR) (TAGGGGTGAAAACGG; SEQ ID NO: 23).The transposon sequence is shown in FIG. 15 (SEQ ID NO: 10). Thems26-ref genomic sequence in its entirety is shown in FIG. 16, SEQ IDNO: 11. A variant of the ms26-ref allele was also found. Sequenceanalysis of this allele, designated ms26′-0406, was found to have lostthe 1430 bp segment found in the last exon of the ms26-ref allele butleft an 8 bp footprint at the site of insertion. Plants homozygous forthe ms26′-0406 allele were male sterile. A comparison of the excisionallele, ms26′-0406 (SEQ ID NO: 8) with the region in the wild-type Ms26gene (SEQ ID NO: 9) is shown in FIG. 14.

Example 4 Expression Analysis and cDNA Isolation

Northern analysis can be used to detect expression of genescharacteristic of anther development at various states ofmicrosporogenesis. Northern analysis is also a commonly used techniqueknown to those skilled in the art and is similar to Southern analysisexcept that mRNA rather than DNA is isolated and placed on the gel. TheRNA is then hybridized with the labeled probe. Potter, E., et al.,“Thyrotrotropin Releasing Hormone Exerts Rapid Nuclear Effects toIncrease Production of the Primary Prolactin in RNA Transcript,” Proc.Nat. Acad. Sci. USA 78:6662-6666 (1981), Lechelt, et al., “Isolation &Molecular Analysis of the Plows,” Mol. Gen. Genet. 219:225-234 (1989).The PstI fragment from the SBMu200-3.1 clone was used to probe aNorthern blot containing kernel, immature ear, seedling and tassel RNA.A signal was seen only in tassel RNA at approximately the quartet stageof microsporogenesis, as reflected in FIG. 3. The transcript is about2.3 kb in length. The same probe was also used to screen a cDNA libraryconstructed from mRNA isolated from meiotic to late uninucleate stagedanthers. One clone, designated Ms26-8.1, was isolated from the library.

Example 5 Sequence and Expression Analysis

The SBMu200-3.1 genomic clone and the Ms26-8.1 cDNA clone were sequencedby Loftstrand Labs Limited. Sanger, F., Nicklen, S., Coulson A. R.(1977) “DNA sequencing with chain terminating inhibitors” Proc. Natl.Acad. Sci. USA 74:5463-5467. The sequences are set forth in FIGS. 4 and5 and the comparison is at FIG. 6. The cDNA/genomic comparison revealsfive introns are present in the genomic clone. The Mu8 insertion occursin exon 1. Testing for codon preference and non-randomness in the thirdposition of each codon was consistent with the major ORF in the cDNAbeing the likely protein-coding ORF. There is a putative Met start codonat position 1089 in the genomic clone. The cDNA homology with respect tothe genomic clone begins at nucleotide 1094. Thus Ms26-8.1 does notrepresent a full length clone and lacks 5 bases up to the putative Metstart codon. A database search revealed significant homology to P450enzymes found in yeast, plants and mammals. P450 enzymes have beenwidely studied and three characteristic protein domains have beenelucidated. The Ms26 protein contains several structural motifscharacteristic of eukaryotic P450's, including the heme-binding domainFxxGxRxCxG (domain D; SEQ ID NO: 24), domain A A/GGXD/ETT/S(dioxygen-binding), domain B (steroid-binding), and domain C. The highlyconserved heme-binding motif was found in MS26 as FQAGPRICLG (SEQ ID NO:25), 51 amino acids away from C-terminus. The dioxygen binding domainAGRDTT (SEQ ID NO: 35) was located between amino acids 320-325. Thesteroid-binding domain was found as LVYLHACVTETLR (SEQ ID NO: 27), aminoacids 397-409. The most significant homologous sequence detected inGenebank database is a deduced protein sequence from rice (GeneBankaccession number 19071651). The second highest homologous sequence is aputative Arabidopsis P450 gene (CYP704B1) whose function is alsounknown. FIG. 17A shows a sequence alignment between CYP704B1 (SEQ IDNO: 12) and Ms26 (SEQ ID NO: 13). Phylogenetic tree analysis of someP450 genes revealed that Ms26 is most closely related to P450s involvedin fatty acid omega-hydroxylation found in Arabidopsis thaliana andVicia sativa (FIG. 17B). The translational frame shift caused in thems26′-0406 excision mutation is believed to destroy the activity of theheme binding domain, thus resulting in sterility. See the comparison atFIG. 18 (Ms26 cDNA at SEQ ID NO: 14; fertile exon 5 region at SEQ ID NO:15 and sterile exon 5 region is SEQ ID NO: 16).

Further expression studies were done using the Ms26 cDNA probe against anorthern containing mRNA at discrete stages of microsporogenesis. FIG.7A shows a Northern blot with RNA samples from different tissuesincluding root (1), leaf (2), husk (3), cob (4), ear spikelet (5), silk(6), immature embryo (7) mature embryo (8), and tassel from, fertileplant (9), ms26-m2::Mu8 sterile plant (10), ms26-ref sterile plant (11)and fertile plant (12). A hybridization signal using Ms26 cDNA wasdetected only in tassel tissues. FIG. 7B shows a Northern blotcontaining mRNA at discrete stages of microsporogenesis. Hybridizationsignals using Ms26 cDNA were detected from meiosis II/quartet stage (4)to late-uninucleate stage (10), with the maximal signal being observedfrom early-uninucleate through late-uninucleate stage (10).

Example 6 Identification of Promoter and its Essential Regions

A putative TATA box can be identified by primer extension analysis asdescribed in by Current Protocols in Molecular Biology, Ausubel, F. M.et al. eds; John Wiley and Sons, New York pp. 4.8.1-4.8.5 (1987).

Regulatory regions of anther genes, such as promoters, may be identifiedin genomic subclones using functional analysis, usually verified by theobservation of reporter gene expression in anther tissue and a lowerlevel or absence of reporter gene expression in non-anther tissue. Thepossibility of the regulatory regions residing “upstream” or 5′ ward ofthe translational start site can be tested by subcloning a DNA fragmentthat contains the upstream region into expression vectors for transientexpression experiments. It is expected that smaller subgenomic fragmentsmay contain the regions essential for male-tissue preferred expression.For example, the essential regions of the CaMV 19S and 35S promotershave been identified in relatively small fragments derived from largergenomic pieces as described in U.S. Pat. No. 5,352,605.

The selection of an appropriate expression vector with which to test forfunctional expression will depend upon the host and the method ofintroducing the expression vector into the host and such methods arewell known to one skilled in the art. For eukaryotes, the regions in thevector include regions that control initiation of transcription andcontrol processing. These regions are operably linked to a reporter genesuch as UidA, encoding-glucuronidase (GUS), or luciferase. Generaldescriptions and examples of plant expression vectors and reporter genescan be found in Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology; Glick, et al. eds;CRC Press; pp. 89-119; (1993). GUS expression vectors and GUS genecassettes are commercially available from Clonetech, Palo Alto, Calif.,while luciferase expression vectors and luciferase gene cassettes areavailable from Promega Corporation, Madison, Wis. Ti plasmids and otherAgrobacterium vectors are described in Ishida, Y., et al., NatureBiotechnology; Vol. 14; pp. 745-750; (1996) and in U.S. Pat. No.5,591,616 “Method for Transforming Monocotyledons” (1994).

Expression vectors containing putative regulatory regions located ingenomic fragments can be introduced into intact tissues such as stagedanthers, embryos or into callus. Methods of DNA delivery includemicroprojectile bombardment, DNA injection, electroporation andAgrobacterium-mediated gene transfer (see Gruber, et al., “Vectors forPlant Transformation,” in Methods in Plant Molecular Biology andBiotechnology, Glick, et al. eds.; CRC Press; (1993); U.S. Pat. No.5,591,616; and Ishida, Y., et al., Nature Biotechnology; Vol. 14; pp.745-750; (1996)). General methods of culturing plant tissues are foundin Gruber, et al., supra and Glick, supra.

For the transient assay system, staged, isolated anthers are immediatelyplaced onto tassel culture medium (Pareddy, D. R. and J. F. Petelino,Crop Sci. J.; Vol. 29; pp. 1564-1566; (1989)) solidified with 0.5%Phytagel (Sigma, St. Louis) or other solidifying media. The expressionvector DNA is introduced within 5 hours preferably bymicroprojectile-mediated delivery with 1.2 μm particles at 1000-1100Psi. After DNA delivery, the anthers are incubated at 26° C. upon thesame tassel culture medium for 17 hours and analyzed by preparing awhole tissue homogenate and assaying for GUS or for lucifierase activity(see Gruber, et al., supra).

Upstream of the likely translational start codon of Ms26, 1088 bp of DNAwas present in the genomic clone ms26-m2::Mu8. Translational fusions viaan engineered NcoI site were generated with reporter genes encodingluciferase and β-glucuronidase to test whether this fragment of DNA hadpromoter activity in transient expression assays of bombarded planttissues. Activity was demonstrated in anthers and not in coleoptiles,roots and calli, suggesting anther-preferred or anther-specific promoteractivity.

A reasonable TATA box was observed by inspection, about 83-77 bpupstream of the translational start codon. The genomic clonems26-m2::Mu8 thus includes about 1005 bp upstream of the possible TATAbox. For typical plant genes, the start of transcription is 26-36 bpdownstream of the TATA box, which would give the Ms26 mRNA a5′-nontranslated leader of about 48-58 nt. The total ms26-m2::Mu8subgenomic fragment of 1088 bp, including nontranslated leader, start oftranscription, TATA box and sequences upstream of the TATA box, was thusshown to be sufficient for promoter activity. See FIG. 8, which is SEQ.ID N0.5. The putative TATA box (TATATCA) is underlined. Thus, thepresent invention encompasses a DNA molecule having a nucleotidesequence of SEQ ID NO: 5 (or those with sequence identity) and havingthe function of a male tissue-preferred regulatory region.

Deletion analysis can occur from both the 5′ and 3′ ends of theregulatory region: fragments can be obtained by site-directedmutagenesis, mutagenesis using the polymerase chain reaction, and thelike (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)).The 3′ end of the male tissue-preferred regulatory region can bedelineated by proximity to the putative TATA box or by 3′ deletions ifnecessary. The essential region may then be operably linked to a corepromoter of choice. Once the essential region is identified,transcription of an exogenous gene may be controlled by the maletissue-preferred region of Ms26 plus a core promoter. The core promotercan be any one of known core promoters such as a Cauliflower MosaicVirus 35S or 19S promoter (U.S. Pat. No. 5,352,605), Ubiquitin (U.S.Pat. No. 5,510,474), the 1N2 core promoter (U.S. Pat. No. 5,364,780), ora Figwort Mosaic Virus promoter (Gruber, et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology;Glick, et al. eds.; CRC Press; pp. 89-119; (1993)). Preferably, thepromoter is the core promoter of a male tissue-preferred gene or theCaMV 35S core promoter. More preferably, the promoter is a promoter of amale tissue-preferred gene and in particular, the Ms26 core promoter.

Further mutational analysis, for example by linker scanning, a methodwell known to the art, can identify small segments containing sequencesrequired for anther-preferred expression. These mutations may introducemodifications of functionality such as in the levels of expression, inthe timing of expression, or in the tissue of expression. Mutations mayalso be silent and have no observable effect.

The foregoing procedures were used to identify essential regions of theMs26 promoter. After linking the promoter with the luciferase markergene deletion analysis was performed on the regions of the promoterupstream of the putative TATA box, as represented in FIG. 9. The x-axisof the bar graph indicates the number of base pairs immediately upstreamof the putative TATA box retained in a series of deletion derivativesstarting from the 5′ end of the promoter. The y-axis shows thenormalized luciferase activity as a percent of full-length promoteractivity.

As is evident from the graph, approximately 176 bp immediately upstreamof the TATA box was sufficient, when coupled to the core promoter(putative TATA box through start of transcription), plus 5′nontranslated leader, for transient expression in anthers. By contrast,luciferase activity was minimal upon further deletion from the 5′ end to91 bp upstream of the putative TATA box. This 176 bp upstream of theputative TATA box through the nontranslated leader can be considered aminimal promoter, which is further represented at FIG. 10. The TATA boxis underlined. Deletion within the full-length promoter from −176through −92 relative to the TATA box reduced activity to about 1% ofwild type. Deletion of −39 through −8 did not greatly reduce activity.Therefore the −176 to −44 bp region contains an essential region andthus would constitute an upstream enhancer element conferring antherexpression on the promoter, which we refer to as an “anther box”.

Linker scanning analysis was conducted across the anther box in 9-10 bpincrements. The locations of the linker scanning substitutions in thisregion are shown in FIG. 10, and the expression levels of the mutantsrelative to the wild type sequence are shown in FIG. 11. The mostdrastic effect on transient expression in anthers was observed formutants LS12 and LS13, in the region 52-71 bp upstream of the putativeTATA box. A major effect on transient expression in anthers was alsoobserved for mutants LS06, LS07, LS08 and LS10, within the region 82-131bp upstream of the putative TATA box. Sequences within the anther boxrequired for wild type levels of transient expression in anthers arethus demonstrated in the −52 to −131 region relative to the putativeTATA box, particularly the −52 to −71 region. The essential regions areshown at SEQ ID NO: 6 (FIG. 10) and, as compared to the genomicsequence, SEQ ID NO: 7 (FIG. 5) are bases 1-1088; 830-962; 830-914;917-962; 875-954; 935-954; and 875-924.

Example 7 Ms26 Sorghum, Rice and Maize Comparison

As noted above, Ms26 is a male fertility gene in maize. When it ismutated, and made homozygous recessive, male sterility will result. Anorthologue of Ms26 was identified in sorghum. The sorghum orthologue ofthe Ms26 cDNA was isolated by using the maize Ms26 gene primers in apolymerase chain reaction with sorghum tassel cDNA as the template. Theresultant cDNA fragment was sequenced by methods described supra andthen compared to the Ms26 cDNA from maize. Nucleotide sequencecomparisons are set forth in FIG. 12 and show 90% identity. Anorthologue from rice was also identified and the predicted codingsequence (SEQ ID NO: 17) and protein (SEQ ID NO: 18) is set forth inFIG. 19. It has one intron less than the maize and sorghum Ms26, and thecoding sequences are highly conserved.

Identification of the sorghum and rice promoters was accomplished. FIG.20 shows an alignment of the Ms26 promoter of corn (SEQ ID NO: 5),sorghum (SEQ ID NO: 19) and rice (SEQ ID NO: 20). The last three basesof the corn promoter shown in the figure is the ATG start oftranslation.

Alignment as reflected in FIG. 21 of the maize Ms26 protein (SEQ ID NO:2), rice Ms26 protein (SEQ ID NO: 18) and sorghum Ms26 protein (SEQ IDNO: 4), and a consensus sequence (SEQ ID NO: 21). The comparison ofprotein sequences shows the protein is highly conserved among theorthologues, with the rice protein sharing 92% similarity and 86%identity when compared to the maize orthologue. The predicted tissuespecificity in rice and sorghum is further reflected in a comparison ofthe Ms26 protein in the sorghum and rice EST database derived frompanicle (flower) libraries. Sorghum sequences producing significantalignments (GenBank accession numbers BI075441.1; BI075273.1;BI246000.1; BI246162.1; BG948686.1; BI099541.1 and BG948366.1, amongothers) all were sequences from immature panicle of sorghum, andsequences showing significant alignment in rice (GenBank accessionnumbers C73892.1; CR290740.1, among others) were also from rice immaturepanicle.

As is evident from the above, nucleotide sequences which map to theshort arm of chromosome 1 of the Zea mays genome, at the same site asthe Ms26 gene, ms26-m2::Mu8 and its alleles, are genes critical to malefertility in plants, that is, are necessary for fertility of a plant,or, when mutated from the sequence found in a fertile plant, causesterility in the plant.

Example 8 Construction of a Plant Transformation Vector Comprising aSelectable Marker, a Male Fertility Gene Ms45 and a Pollen CytotoxinGene

A construct designated PHP18091, shown in FIG. 22 is made by assemblingfollowing DNA components:

-   1. The plasmid pSB 11 backbone DNA (pSB31 lacking the EcoRI fragment    carrying the 35SGUS and 35SBAR genes, Ishida et al., Nature    Biotechnol. (1996) 14:745-750). This DNA backbone contains T-DNA    border sequences and the replication origin from pBR322.-   2. The 35S:PAT gene which encodes the enzyme phosphinothricin    acetyltransferase (PAT) from Streptomyces viridochomagenes    (nucleotides 6-557 from accession number A02774, Strauch et al.    1988, EP 0275957-A) under the transcriptional control of the    cauliflower mosaic virus (CaMV) 35S promoter and terminator    (nucleotides 6906-7439, and 7439-7632, respectively from Franck et    al. 1980, Cell 21: 285-294).-   3. The 5126:Ms45 gene which contains the maize male fertility gene    coding region (nucleotides 1392-3343, accession number AF360356,    Albertsen et a Am. J. Bot. (1993) 80:16) under the control of the    maize anther-specific promoter 5126 (nucleotides 985-1490, accession    number 175204).-   4. The PG47:DAM gene which contains the E. coli DNA (Adenosine-N⁶)    methyltransferase (DAM) coding region (nucleotides 195-1132, Brooks    et al., Nucleic. Acids Res (1983) 11: 837-851) driven by the maize    pollen-specific promoter PG47 (nucleotides 1-2870, accession number    X66692, Allen and Lonsdale, Plant J. (1993) 3:261-271). The    transcription of this gene is terminated by the potato proteinase    inhibitor II (PinII) terminator (nucleotides 2-310, An et al., Plant    Cell (1989) 1: 115-122).-   5. A 3.34 kb NcoI DNA fragment containing Ms45:Ms45 was cloned    upstream of the 35S:PAT gene in pUC8, creating PHP6641. A 4.7 kb    HindIII/EcoRI DNA fragment containing Ms45:Ms45-35S:PAT from PHP6641    was cloned into pSB11, creating PHP10890 (Cigan et al, Sex. Plant    Reprod. (2001) 14: 135-142). The native Ms45 promoter in PHP10890    was replaced by a 528 bp HindIII/NcoI fragment containing the maize    5126 promoter, creating PHP11943.-   6. A 2.87 kb HindIII/NcoI fragment containing PG47 promoter was    ligated with a 0.8 kb NcoI/HindIII fragment containing the DAM    coding region, PinII terminator and 35S enhancer which was from    PHP10404 (Unger, et al., Transgenic Res. (2001) 10: 409-422),    creating a 3.67 kb fragment HindIII fragment containing PG47:DAM    gene fusion (with the 35S enhancer). This 3.67 kbp HindIII fragment    was then cloned into the HindIII site of PHP11943, creating    PHP20005. The 35S enhancer in PHP20005 was removed, creating    PHP18071. The PHP18071 was introduced into Agrobacterium strain    LBA4404 carrying plasmid pSB 1 by triparental mating (Ishida et al.,    Nature Biotechnol. (1996) 14:745-750). The co-integrate of PHP18071    and pSB1 was named PHP18091.

Example 9 Transformation of Corn with the Restoring Transgene Constructof Example 8

A male-sterile female which was homozygous for an ms45 mutant Acexcision allele, ms45′-9301 (ms45) was repeatedly crossed with bulkedpollen from maize Hi-type II plants (Armstrong 1994, In: Freeling andWalbot (eds). The Maize Handbook. Springer, N.Y., pp 663-671) resultingin the introgression of this ms45 allele in transformation amenablemaize germplasm over multiple generations. The resultant source ofmaterial for transformation consisted of embryos segregating (1:1 or3:1) for ms45 and allowed for both transformation directly into ahomozygous ms45 background and to test the genetic complementation ofthe ms45 mutation in T₀ plants. Agrobacterum-mediated transformation wasperformed according to Zhao et al. 1999, (U.S. Pat. No. 5,981,840).Genotyping and molecular analysis (integration and PTU) of transformantswere done according Cigan et al., (Sex. Plant. Reprod. (2001)14:135-142). Transformants with single-integration and complete PTU wereselected for further studies.

Example 10 Analysis of Maize Transformants

Transgenic plants (T₀) from Example 9 were evaluated for the whole plantmorphology and analyzed for transgene transmission through both pollenand egg cells. No morphological difference was observed between thetransgenic plants and the non-transgenic control plants except for thedegree of male fertility. Transformants with single-integration andintact PTU were partial male fertile while non-transgenic control plantswere completely male sterile, indicating that the expression of Ms45gene complemented the homozygous recessive ms45 male sterile phenotype.This also demonstrated that the expression of the DAM gene causedpartial male sterility by eliminating the pollen grains carrying thetransgenes. Without the DAM gene, Ms45 transgene can completely recoverthe ms45 male sterile mutation (Cigan et al., Sex. Plant. Reprod. (2001)14: 135-142). The correct function of DAM gene was further determined bycontrolled pollinations between T₀ transgenic plants and non-transgenicplants. Pollen grains from T₀ transgenic plants were used to pollinatenon-transgenic plants control plants. Immature embryos were harvestedfrom ears of these non-transgenic plants 18 days after pollination andcultured either on MS media or MS media containing 3.0 mg/L of bialaphos(Murashige, T. and Skoog, F. A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol. Plant (1962) 15:437-439). 100% of the embryos were able to germinate on control mediumwhile none of the embryos were able to germinate on media containing 3mg/L of bialaphos, indicating that the restoring transgene construct wasnot transmitted through pollen to progeny.

In addition, pollen from non-transgenic plants was used to pollinate theT₀ transgenic maintainer plants. Immature embryos were harvested fromears of these T₀ transgenic maintainer plants 18 days after pollinationand cultured as above control media or media containing 3 mg/L ofbialaphos. All embryos were able to germinate on control medium while50% of the embryos were able to germinate on the medium containingbialaphos, indicating that the restoring transgene construct wastransmitted through the ovule to progeny at the expected frequency. Theresults of embryo rescues are summarized in Tables 1 and 2.

TABLE 1 Transgene transmission through pollen Pollen to non-transgenicplants Control medium Medium + 3 mg/l bialaphos # # Transgenic embryo #embryo embryo # embryo plants cultured germinated % cultured germinated% 14089263 40 40 100 60 0 0 14089277 100 100 100 100 0 0 14089839 40 40100 60 0 0

TABLE 2 Transgene transmission through egg cells Pollen fromnon-transgenic plants Medium + 3 mg/l bialaphos Transgenic # embryo #embryo plants cultured germinated % 14089262 20 8 40 14089277 40 22 5514089839 40 21 53

Example 11 Conversion of T₀ Plants into Different Inbred Lines andAnalysis of Tn Plants

T₀ transgenic maintainer plants from Example 9 were converted intodifferent inbred backgrounds through repeated backcross by pollinationfrom inbred lines such as PH09B. To accomplish this, pollen produced byPH09B that is ms45 heterozygous background were used to pollinate theears of T₀ maintainer plants that were homozygous for the ms45 mutantalleles. T₁ seed harvested from these T₀ plants segregated for bothtransgenes and ms45 alleles. T₁ plants that did not contain therestoring transgene construct were eliminated by herbicide selection. T₁plants containing transgenes were analyzed for ms45 background and malefertility according to Cigan et al., (Sex. Plant. Reprod., (2001) 14:135-142). In general, T₁ plants in homozygous ms45 condition thatcontained the restoring transgene construct showed partial malefertility like that observed for the T₀ parent plants, while the T₁plants in homozygous ms45 condition but containing no transgenes werecomplete male sterile. This suggested that the Ms45 transgene continuedto function correctly in a different genetic background. Pollen grainsfrom T₁ plants were examined for viability using microscopic andhistochemical staining. Pollen grains at different developmental stageswere collected and stained with fluorescein diacetate (FDA), 4′,6-diamidino-2-phenylindole (DAPI) and ethidium bromide (EB). About 50%of the pollen grains from the transgenic T₁ plants lost their viabilityas judged by the absence of fluorescence after staining with FDA afterfirst pollen mitosis, while the pollen grains from non-transgeniccontrol plants showed uniform FDA staining. This was further supportedby in vitro pollen germination studies. The germination rate of thepollen grains from the transgenic T₁ plants were about half of that fromnon-transgenic control plants. Pollen grains from transgenic T₁ plantwere also used to pollinate non-transgenic plants to test transgenetransmission thought pollen. For instance, none of 248 embryos from anon-transgenic plant pollinated by a T₁ plant (20118954) were able togerminate on the medium containing 3 mg/l bialaphos. These experimentsconfirmed both the correct function of the Ms45 and DAM transgenes indifferent genetic backgrounds. The T₁ plants with desired performancewere used for the next backcross iteration using pollen from thepaternal inbred parent which was heterozygous for the mutant ms45allele. This process will be repeated until sixth generation.

Example 12 Large Scale Transmission and Maintenance of ms45 MaleSterility Using the Construct of Example 8

T₁ plants derived from T₀ 14089277 as described in example 9 were usedas males to pollinate either wild type inbred plants or ms45/ms45 malesterile inbred plants. The 10,117 T₂ progeny from the wild type crossesand 6688 T₂ progeny from the ms45/ms45 crosses were evaluated fortransgene transmission by screening for herbicide resistance. For bothtypes of crosses a total of 16786 T₂ plants were found to be herbicidesensitive, yielding a non-transmission frequency of 99.89%. All T₂plants from the ms45/ms45 crosses that did not contain the transgene,were completely male sterile, indicating that this transgenic line canmaintain ms45 sterility.

Example 13 Construction of a Plant Transformation Vector Comprising aScreenable Marker, a Male Fertility Gene Ms26 and a Pollen CytotoxinGene

A construct designated PHP24101, shown in FIG. 23, is made by assemblingfollowing DNA components:

-   1. The plasmid pSB11 backbone DNA (pSB31 lacking the EcoRI fragment    carrying the 35SGUS and 35SBAR genes, Ishida et al., Nature    Biotechnol. (1996) 14:745-750). This DNA backbone contains T-DNA    border sequences and the replication origin from pBR322.-   2. The PG47PRO:ZM-AA1 gene which contains alpha-amylase 1 coding    region from Zea mays as set forth in FIG. 24. (SEQ ID NO: 26). The    transcription of this gene is terminated by 1N²-1 terminator (U.S.    Pat. No. 5,364,780).-   3. The Ms26 (SB200) GENOMIC gene (SEQ ID NO: 7) which contains the    maize male fertility gene coding region.-   4. LTP2:DS-RED2 (ALT1) which contains red florescence coding region    (a variant of Discosoma sp. red fluorescent protein (DsRed), from    Clontech mutated to remove BstEII site, codon sequence unchanged)    driven by LTP2 promoter, supra.-   5. A 2.143 kb EcoRV/DraI DNA fragment containing LTP2PRO:DS-RED2    (ALT1) from PHP21737 was cloned into downstream of the Ms26 GENOMIC    gene in SK vector, creating SK-Ms26 GENOMIC-LTP2PRO:DS-RED2 (ALT1).-   6. A 2.143 kb EcoRV/DraI DNA fragment containing LTP2PRO:DS-RED2    (ALT1) from PHP21737 was cloned into downstream of the Ms45PRO:Ms45    GENOMIC gene in SK vector, creating SK-Ms45-LTP2PRO:DS-RED2 (ALT1).-   7. A 5.429 kb NotI fragment containing 5126PRO:Ms45    GENOMIC-UBI:MOPAT:PINII in PHP20532 was replaced by A 4.318 kb NotI    fragment containing Ms45-LTP2PRO:DS-RED2 (ALT1) from    SK-Ms45-LTP2PRO:DS-RED2 (ALT1), creating PHP22623.-   8. A 4.318 kb NotI fragment containing Ms45-LTP2PRO:DS-RED2 (ALT1)    in PHP22623 was replaced by A 5.960 kb NotI DNA fragment containing    Ms26 GENOMIC-LTP2PRO:DS-RED2(ALT1) from SK-Ms26    GENOMIC-LTP2PRO:DS-RED2 (ALT1), creating PHP24014. The PHP24014 was    introduced into Agrobacterium strain LBA4404 carrying plasmid pSB1    by Electrophoresis. Co-integrate of PHPPHP24014 and pSB1 was named    PHP24101.

Example 14 Transformation of Corn with the Restoring Transgene Constructof Example 13

A male-sterile female which was homozygous for a ms26 mutant excisionallele, (ms26) was repeatedly crossed with bulked pollen from maizeHi-type II plants (Armstrong 1994, In: Freeling and Walbot (eds). TheMaize Handbook. Springer, N.Y., pp 663-671) resulting in theintrogression of this ms26 allele in transformation amenable maizegermplasm over multiple generations. The resultant source of materialfor transformation consisted of embryos segregating (1:1 or 3:1) forms26 and allowed for both transformation directly into a homozygous ms26background and to test the genetic complementation of the ms26 mutationin T₀ plants. Agrobacterum-mediated transformation was performedaccording to Zhao et al. 1999, (U.S. Pat. No. 5,981,840). Genotyping andmolecular analysis (integration and PTU) of transformants were doneaccording Cigan et al., (Sex. Plant. Reprod. 1 (2001) 4:135-142).Transformants with single-integration and complete PTU were selected forfurther studies.

Example 15 Analysis of Maize Transformants

Transgenic plants (T₀) from Example 14 were evaluated for the wholeplant morphology and analyzed for transgene transmission through pollen.No morphological difference was observed between the transgenic plantsand the non-transgenic control plants except for the degree of malefertility. Transformants with single-integration and intact PTU werepartial male fertile while non-transgenic control plants were completelymale sterile, indicating that the expression of the Ms26 genecomplemented the homozygous recessive ms26 male sterile phenotype. Thisalso suggested that the pollen expression of the alpha amylase (AA) genecaused partial male sterility by disrupting the normal function of thepollen grains carrying the transgenes. Staining pollen fromtransformants with potassium iodide (KI), which stains starch granules,showed that approximately half of the pollen grains contained starch(black grains, non-transgenic) and the other half did not contain starch(gold grains, transgenic). The correct function of AA gene was furtherdetermined by controlled pollinations between T₀ transgenic plants andnon-transgenic plants. Resultant T₁ kernels were evaluated for a redfluorescence phenotype. If the transgenes were transmitted through thepollen then the T₁ seed would contain red fluorescent kernels due to theexpression of RFP in the aleurone layer. For four independent eventsshown in Table 3, no RFP expression was found in the T₁ seed, whereasseed from the T₀ ears themselves (T₁ seed) contained approximately 50%red fluorescent kernels.

TABLE 3 Transgene transmission through pollen Pollen to non-transgenicplants Kernel Red Fluoresence Transgenic plants # Yellow Kernels # RedKernels % 42772379 338 0 100 42772385 277 0 100 42772400 268 0 10042772411 598 0 100

Thus it can be seen that the invention achieves at least all of itsobjectives.

1. A method of preventing formation or function of male tissue in aplant comprising introducing into said plant an expression cassettecomprising a male tissue preferred regulatory region operably linked toa nucleic acid molecule comprising a sequence selected from the groupconsisting of: a) a sequence having at least 80% identity to the fulllength of SEQ ID NO: 26; b) a sequence having at least 85% identity tothe full length of SEQ ID NO: 26; c) a sequence having at least 90%identity to the full length of SEQ ID NO: 26; d) a sequence having atleast 95% identity to the full length of SEQ ID NO: 26; e) a sequencewhich hybridizes to the full length of the complement of SEQ ID NO: 26;f) a sequence which encodes the polypeptide of SEQ ID NO: 36, whereinsaid sequence encodes a polypeptide that prevents formation or functionof male tissue in said plant.
 2. The method of claim 1, wherein saidnucleic acid molecule has at least 80% identity to the full length ofSEQ ID NO:
 26. 3. The method of claim 1, wherein said nucleic acidmolecule has at least 85% identity to the full length of SEQ ID NO: 26.4. The method of claim 1, wherein said nucleic acid molecule has atleast 90% identity to the full length of SEQ ID NO:
 26. 5. The method ofclaim 1, wherein said nucleic acid molecule has at least 95% identity tothe full length of SEQ ID NO:
 26. 6. The method of claim 1, wherein saidnucleic acid molecule comprises SEQ ID NO:
 26. 7. The method of claim 1,wherein said nucleic acid molecule comprises a sequence which hybridizesto the full length of the complement of SEQ ID NO:
 26. 8. The method ofclaim 1, wherein said nucleic acid molecule encodes the polypeptide ofSEQ ID NO:
 36. 9. The method of claim 1, wherein said regulatory regionis selected from the group consisting of the 5126 promoter, the Ms45promoter, the BS92-7 promoter, the Ms26 promoter, and the SGB6 promoter